[go: up one dir, main page]

US20250002873A1 - Retinoid production - Google Patents

Retinoid production Download PDF

Info

Publication number
US20250002873A1
US20250002873A1 US18/700,931 US202218700931A US2025002873A1 US 20250002873 A1 US20250002873 A1 US 20250002873A1 US 202218700931 A US202218700931 A US 202218700931A US 2025002873 A1 US2025002873 A1 US 2025002873A1
Authority
US
United States
Prior art keywords
retinol
host cell
retinoids
mix
retinal
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
US18/700,931
Inventor
Valmik Kanubhai VYAS
Peter Louis HOUSTON
Jenna MCMAHON
René Marcel De Jong
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
DSM IP Assets BV
Original Assignee
DSM IP Assets BV
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by DSM IP Assets BV filed Critical DSM IP Assets BV
Priority to US18/700,931 priority Critical patent/US20250002873A1/en
Assigned to DSM IP ASSETS B.V. reassignment DSM IP ASSETS B.V. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: de Jong, René Marcel, HOUSTON, Peter Louis, MCMAHON, Jenna, VYAS, Valmik Kanubhai
Publication of US20250002873A1 publication Critical patent/US20250002873A1/en
Pending legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
    • C12N15/815—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts for yeasts other than Saccharomyces
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/045—Hydroxy compounds, e.g. alcohols; Salts thereof, e.g. alcoholates
    • A61K31/07—Retinol compounds, e.g. vitamin A
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P23/00—Preparation of compounds containing a cyclohexene ring having an unsaturated side chain containing at least ten carbon atoms bound by conjugated double bonds, e.g. carotenes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C12P7/22—Preparation of oxygen-containing organic compounds containing a hydroxy group aromatic
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/62—Carboxylic acid esters
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
    • C12Y101/01—Oxidoreductases acting on the CH-OH group of donors (1.1) with NAD+ or NADP+ as acceptor (1.1.1)
    • C12Y101/01105—Retinol dehydrogenase (1.1.1.105)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/645—Fungi ; Processes using fungi
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/645—Fungi ; Processes using fungi
    • C12R2001/72—Candida
    • C12R2001/73—Candida lipolytica
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/645—Fungi ; Processes using fungi
    • C12R2001/78—Hansenula

Definitions

  • the present invention is related to bio-production of retinoids with improved purity profile, particularly bio-retinoids with a high percentage of retinyl acetate, wherein the flux towards retinol is increased and wherein the percentage of unwanted by-products such as dihydroretinoids is minimized to a range of below 10%.
  • Retinol is an important intermediate/precursor in the process of retinoid production, particularly such as vitamin A production.
  • Retinoids including vitamin A, are one of very important and indispensable nutrient factors for human beings which have to be supplied via nutrition. Retinoids promote well-being of humans, inter alia in respect of vision, the immune system and growth.
  • retinoids Unfortunately, the biological systems that produce retinoids are industrially intractable and/or produce the compounds at such low levels that commercial scale isolation is not practical. The most limiting factors include instability of intermediates including instability of retinol, inefficient enzymatic conversions and/or impurity due to accumulation of unwanted side-products.
  • RDHs retinal reducing enzymes
  • the present invention is related to a process for production of a retinoid-mix, such as a fermentation process using a suitable retinoid-producing host cell, said retinoid-mix comprising dihydroretinoids with a percentage of less than about 10% based on total retinoids within said mix.
  • a “retinoid mix” is defined as all retinoids formed during a retinoid production process, such as e.g. fermentation process using a suitable host cell that is expressing suitable enzymes involved in formation of retinoids including but not limited to retinal, retinol, and retinyl acetate and optionally furthermore expressing enzymes involved in formation of beta-carotene.
  • Said retinoid mix comprises retinal, retinol, retinyl acetate, and dihydroretinods.
  • such retinoid-mix comprises dihydroretinoids, retinal, and retinyl acetate, wherein the percentage of dihydroretinoids is less than about 10% based on total retinoids, preferably wherein the percentage of retinal is less than about 8% based on total retinoids, further preferably wherein the percentage of retinyl acetate is at least about 80% based on total retinoids, even further preferably wherein the percentage of trans-isomers within said mix is at least about 80% based on the amount of all cis- and trans-retinoids.
  • the percentage of dihydroretinoids based on total retinoids within said retinoid-mix is about less than 10, such as 9, 8, 7, 6, 5, 4, 3, 1% or less, preferably 2% or less, more preferably 1% or less, such as 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1% or less, such as in the range of 1% to 0.1, 0.01, or 0.001%, as measurable at the end of the production or fermentation process.
  • the percentage of retinal based on total retinoids within said retinoid-mix is about less than 8, such as 7, 6, 5, 4, 3, 1% or less, preferably 2% or less, more preferably 1% or less, such as 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1% or less, such as in the range of 1% to 0.1, 0.01, or 0.001%, as measurable at the end of the production or fermentation process.
  • the percentage of retinyl acetate based on total retinoids within said retinoid-mix is at least about 80%, such as e.g. 85, 90, 92, 95, 98% or more retinyl acetate, as measurable at the end of the production or fermentation process using a suitable retinyl-acetate producing host cell.
  • the percentage of trans-isoforms based on the total amount of cis and trans-isomers in said retinoid-mix is at least about 80, 85, 90, 92, 95, 98% or more, as measurable at the end of the production or fermentation process.
  • said mix comprises at least about 80% of trans-retinyl acetate, i.e. 80% of the retinyl acetate in the retinoid-mix is present in the trans-isomer.
  • the present invention is related to a biotechnological process for production of a retinoid-mix as defined herein comprising enzymatic conversion of retinal into retinol in the presence of an RDH22 homolog, particularly obtainable from fungi, as defined herein, particularly comprising cultivation of a suitable host cell, more particularly a retinal-producing host cell, expressing said RDH22 homolog under suitable conditions that allow formation of said retinoid-mix as defined herein.
  • RDH retina reductase
  • retinal reducing enzyme enzyme involved in formation of retinol
  • enzyme involved in conversion of retinal into retinol are used interchangeably herein and refer to enzymes [EC 1.1.1.105] which are involved in the conversion/catalysis of retinal into retinol as well as the back-conversion from retinol to retinal.
  • the formation of dihydroretinoids is reduced to a percentage of less than about 10% detectable in a retinoid-mix formed during a fermentation process using a suitable retinoid-producing host cell and under suitable conditions and the flux towards retinol is increased such that the percentage of retinal present in said retinoid-mix as detectable at the end of the fermentation process is less than about 8% based on total retinoids.
  • the present invention is related to RDH22 homologs as defined herein capable of catalyzing retinal into retinol, wherein the conversion towards retinol is increased, leading to a retinoid-mix with a percentage of less than about 8% retinal based on total retinoids, which can be measured at the end of the fermentation process by known methods, particularly process for production of a retinoid-mix with at least about 80% retinyl acetate as described herein.
  • the present invention is directed to RDH22 homologs obtainable from Yarrowia , preferably Yarrowia lipolytica , comprising YlRDH22 according to SEQ ID NO:1 and enzymes originated from other source organisms or being artificially constructed (based on digital sequence information) but having activity corresponding to the respective Yarrowia RDH22 according to SEQ ID NO: 1.
  • YlRDH22 homolog as defined herein in a process comprising production of retinol and retinyl acetate, the percentage of retinal accumulated during such production process and present in the retinoid-mix measured at the end of the production process could be reduced to less than 1% based on total retinoids.
  • the present invention is directed to polypeptides with at least about 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 92, 95, 97, 98, 99% or up to 100% identity to SEQ ID NO:1 including polynucleotides encoding said polypeptides, such as e.g. polynucleotide according to SEQ ID NO:2 or 3 as well as enzymes having equivalent enzymatic activity, i.e. activity corresponding to YlRDH22 as defined above but are originated from another source organism or being based on digital sequence information, particularly being originated from another fungal organism.
  • the present invention is directed to RDH22 homologs obtainable from Wickerhamomyces , preferably Wickerhamomyces anomalus , comprising WaRDH22 according to SEQ ID NO:4 and enzymes originated from other source organisms or being artificially constructed (based on digital sequence information) but having activity corresponding to the respective Wickerhamomyces RDH22 according to SEQ ID NO:4.
  • WaRDH22 homolog as defined herein in a process comprising production of retinol and retinyl acetate, the percentage of retinal accumulated during such production process and present in the retinoid-mix measured at the end of the production process could be reduced to about 2% or less based on total retinoids. with a conversion rate of more than 90% towards formation of retinol.
  • the present invention is directed to polypeptides with at least about 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 92, 95, 97, 98, 99% or up to 100% identity to SEQ ID NO:4 including polynucleotides encoding said polypeptides, such as e.g. polynucleotide according to SEQ ID NO:5 or 6 as well as enzymes having equivalent enzymatic activity, i.e. activity corresponding to WaRDH22 as defined above but are originated from another source organism or being based on digital sequence information, particularly being originated from another fungal organism.
  • the RDH22 enzymes as defined herein are catalyzing the conversion of retinal into retinol with a conversion being shifted towards production of retinol, wherein the substrate (i.e. retinal) can be either cis-, trans- or a mix of cis-/trans-retinal in any possible ratio, with a percentage of at least about 80% trans-isomers being preferred.
  • retinol and optionally retinyl acetate with about the same ratio of trans to cis-isomers in a respective retinoid-mix obtained by a process for fermentative production as defined herein, such as starting from beta-carotene using a host cell capable of retinal, retinol and/or retinyl-acetate production.
  • beta-carotene oxygenases BCOs
  • Enzymes particularly acetyl transferases (ATFs), more preferably ATF1s, host cells and processes for conversion of retinol into retinyl acetate are known, such as e.g. from WO2019058000 or WO2020141168.
  • conversion “enzymatic conversion or “cleavage” in connection with enzymatic catalysis of substrates such as e.g. beta-carotene, retinal or retinol are used interchangeably herein and refer to the action of the specific enzymes, including but not limited to BCOs, RDHs, particularly RDH22, or ATFs as defined herein involved in formation of retinal, retinol or retinyl acetate.
  • dihydroretinoids and “13,14-dihydroretinoids” are used interchangeably herein and includes but not limited to dihydroretinol, dihydroretinal, dihydroretinyl acetate that can be detected in a retinoid-mix according to the present invention. The skilled person known how to measure this.
  • the present invention is related to a process for reducing the percentage of by-products including e.g. retinal and dihydroretinoids present in a retinoid-mix as defined herein, particularly wherein a retinoid-producing host cell, such as a retinal/retinol-producing host cell is modified leading to (over) expression of enzymes having RDH22 activity, particularly YlRDH22 or WaRDH22 and homologs thereof as defined herein, wherein the reduction of retinal is in the range of 20 to more than 92%, particularly about 25, 30, 40, 50, 70, 75, 80, 85, 90, 92, 95% or more and/or the reduction of dihydroretinoids is in the range of 20 to more than 97%, particularly about 25, 30, 40, 50, 70, 75, 80, 85, 90, 92, 95, 97% or more as compared to a process using a non-modified host cell, i.e. a host cell that has not been transformed with such heterologous RDH22 poly
  • a “modified host cell” refers to a host cell or a process wherein the host cell is (over) expressing a heterologous RDH22 homolog as defined herein, including but not limited to host cells modified such that the activity of an endogenous RDH22 homolog as defined herein is increased as compared to the wild-type or non-modified host cell, such as e.g. via expression of 2, 3, 4, or more gene copies of an endogenous gene, i.e. expression of more copies of the respective endogenous gene as occurring in the respective non-modified wild-type host cell.
  • a modified host cell is also known as “recombinant host cell”.
  • Modifications in order to generate a recombinant host cell having increased activity of an RDH22 homolog as defined herein may include the use of strong promoters, suitable transcriptional- and/or translational enhancers, or the introduction of one or more gene copies into the respective wild-type host cell, leading to increased accumulation of the respective enzymes and the respective products (e.g. retinol, retinyl acetate) in a given time.
  • the skilled person knows which techniques to use in dependence of the host cell.
  • the increase (or reduction) of gene expression can be measured by various methods, such as e.g. Northern, Southern, or Western blot technology as known in the art.
  • novel RDH22 homologs as defined herein can be achieved in any host system, including (micro) organisms, which is suitable for conversion of retinal into retinol (i.e. retinol-producing host cell) and further suitable for conversion of retinol into retinyl acetate (i.e. retinyl-acetate producing host cell) and furthermore suitable for conversion of carotenoid precursors into beta-carotene (i.e. carotenoid-producing host cell) and furthermore suitable for conversion of beta-carotene into retinal (i.e. retinal-producing host cell) and which allows the expression of the enzymes as disclosed herein and as used for such processes.
  • retinol i.e. retinol-producing host cell
  • retinyl acetate i.e. retinyl-acetate producing host cell
  • carotenoid precursors i.e. carotenoid precursors into beta-carotene
  • suitable host cells to be used for the present invention might be selected from bacteria, algae, fungi including yeast, plant or animal cells, such as e.g. fungal host cells including oleaginous yeast cells, such as e.g. Rhodosporidium, Lipomyces, Saccharomyces or Yarrowia , preferably Yarrowia , more preferably Yarrowia lipolytica or bacterial host cells including but not limited to Escherichia or Pantoea .
  • the skilled person knows which genes are suitable in order to express the necessary enzymes involved in carotenoid—and/or retinoid biosynthesis in a suitable host cell. Genes and methods to generate carotenoid-producing host cells are known in the art, see e.g. WO2006102342. Depending on the carotenoid to be produced, different genes might be involved.
  • the term “fungal host cell” particularly includes yeast cells, such as e.g. retinal/retinol/retinyl-acetate-producing yeast cells, comprising Yarrowia or Saccharomyces .
  • yeast cells such as e.g. retinal/retinol/retinyl-acetate-producing yeast cells, comprising Yarrowia or Saccharomyces .
  • retinyl-acetate producing host cells includes host cells, capable of synthesizing retinol and expressing acetyl transferases (ATFs) as defined in e.g. WO2019058001 or WO2020141168 resulting in retinyl acetate with a percentage as defined herein based on total retinoids produced by said host cell.
  • such host cell is furthermore capable of producing carotenoids.
  • the host cell expressing an enzyme having activity of YlRDH22 or WaRDH22 as defined herein, including enzymes with at least about 30% identity to YlRDH22 according to SEQ ID NO:1 or WaRDH22 according to SEQ ID NO:4 to be used for fermentative production of a retinoid-mix according to the present invention comprises further modifications enabling the conversion of retinol into retinyl acetate via action/expression of heterologous ATF [EC 2.3.1.84], 20) particularly fungal ATF, comprising a highly conserved partial amino acid sequence of at least 7 amino acid residues selected from [NDEHCS]-H-x(3)-D-[GA] (motifs are in Prosite syntax, as defined in https://prosite.expasy.org/scanprosite/scanprosite_doc.html), wherein “x” denotes an arbitrary amino acid and with the central histidine being part of the enzyme's binding pocket, preferably wherein the 7 amino acid motif is selected from [NDE]
  • Such enzymes might be particularly selected from L. mirantina, L. fermentati, S. bayanus , or W. anomalus , such as disclosed in WO2020141168 or WO2019058001, more preferably said ATFs comprising one or more amino acid substitution(s) in a sequence with at least about 20%, such as e.g. 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 92, 95, 97, 98, 99% or up to 100% identity to LmATF1 (i.e.
  • SEQ ID NO:1 as disclosed in WO2020141168
  • the one or more amino acid substitution(s) are located at position(s) corresponding to amino acid residue(s) selected from the group consisting of position 68, 69, 72, 73, 171, 174, 176, 178, 291, 292, 294, 301, 307, 308, 311, 312, 320, 322, 334, 362, 405, 407, 409, 480, 483, 484, 490, 492, 520, 521, 522, 524, 525, 526 and combinations thereof and as particularly exemplified in Table 4 of WO2020141168, most preferably comprising one or more amino acid substitution(s) on positions corresponding to amino acid residue(s) 69, 407, 409, 480, 484, and combinations thereof in SEQ ID NO:1 as disclosed in WO2020141168, even more preferably comprising an amino acid substitution at a position corresponding to residue 69 in the polypeptide according to SEQ ID NO: 1 as disclosed in WO
  • H69N histidine by asparagine
  • H69S serine
  • H69A alanine
  • H69A amino acid substitution at a position corresponding to residue 407 in the polypeptide according to SEQ ID NO:1 as disclosed in WO2020141168 leading to isoleucine at said residue
  • V4071 valine by isoleucine
  • an amino acid substitution at a position corresponding to residue 409 in the polypeptide according to SEQ ID NO: 1 as disclosed in WO2020141168 leading to alanine at said residue such as e.g.
  • S480E glutamic acid
  • S480L lysine
  • S480M methionine
  • S480F phenylalanine
  • S480Q glutamine
  • Said modified enzyme might be originated from yeast, such as e.g. L. mirantina, L. fermentati, W. anomalus or S. bayanus , preferably from L. mirantina .
  • the ATF to be used for the process according to the present invention is a modified ATF1 comprising amino acid substitutions S480Q_G409A_V4071_H69A_1484L and is obtainable from Lachancea mirantina , including an enzyme with an identity of at least about 20% to the LmATF1 according to SEQ ID NO:1 as disclosed in WO2020141168.
  • Suitable culture conditions of a fermentation process include cultivation of the host cell in an aqueous medium in the presence of suitable carbon sources as defined herein, optionally supplemented with appropriate nutrients under aerobic or anaerobic conditions and as known by the skilled person to enable production of a retinoid-mix as defined herein.
  • the fermentation may be conducted in batch, fed-batch, semi-continuous or continuous mode. Particularly, fermentations are run in fed-batch stirred tank reactors. Fermentations can be run for 5 to 14 days, such as e.g. for around 118 h. Fermentation products including but not limited to retinol, retinyl acetate may be harvested from the cultivation at a suitable moment, e.g.
  • the retinoid-mix comprising retinol and acetylated retinoids might be further purified and/or further processed/formulated, to be used in the food, feed, pharma or cosmetic industry. Cultivation and isolation of host cells selected from Yarrowia or Saccharomyces to be used for production of carotenoids and/or retinoids is described in e.g. WO2008042338.
  • Carbon sources to be used for the present invention might be selected from linear alkanes, free fatty acids, including triglycerides, particularly vegetable oil, such as e.g. selected from the group consisting of oil originated from corn, soy, olive, sunflower, canola, cottonseed, rapeseed, sesame, safflower, grapeseed or mixtures thereof, including the respective free fatty acids, such as e.g. oleic acid, palmitic acid or linoleic acid.
  • Suitable carbon sources might furthermore be selected from ethanol, glycerol or glucose and mixtures of one or more of the above-listed carbon sources.
  • the present invention is directed to a process for the production of retinoids, in particular a retinoid-mix as defined herein, in a two-phase culture system including an in vitro extraction system, i.e. cultivation in the presence of a lipophilic solvent as defined herein, wherein a retinal-/retinol-/retinyl acetate-producing host cell, preferably oleaginous yeast cell such as e.g. Yarrowia , is cultivated under suitable culture conditions such that the retinoid-mix, including retinyl acetate, is accumulated in the lipophilic solvent and optionally extracted/isolated and/or purified from said lipophilic solvent.
  • a retinal-/retinol-/retinyl acetate-producing host cell preferably oleaginous yeast cell such as e.g. Yarrowia
  • Preferred lipophilic solvents to be used for this aspect of the present invention might be selected from isoparaffins including mixtures of alkanes, cycloparaffin, isoalkanes, cycloalkanes, or dodecanes.
  • the solvents might be natural or synthetic ones.
  • Examples of commercially available useful solvents might be selected from Total, e.g. Isane® solvents, Shell, e.g. ShellSolTD or ShellSolT, Exxon Mobile, e.g. IsoparTM fluids, particularly such as e.g.
  • Isopar M, Isopar N, Isopar H, Isopar K, Isopar L, or mixtures with iso-dodecane isomers as e.g. commercially available under the tradename AC365770010 (Acros Organics).
  • the second phase solvent is selected from isoparaffins, such as e.g. Isopar M, Isopar N, Isopar H, Isopar K, Isopar L, more preferably selected from Isopar L, Isopar M or Isopar N, solvents with equivalent or identical properties but from other suppliers, wherein said solvents are preferably not being consumed or evaporated during the fermentation process, and/or wherein the color of the produced retinyl acetate is transparent.
  • isoparaffins such as e.g. Isopar M, Isopar N, Isopar H, Isopar K, Isopar L, more preferably selected from Isopar L, Isopar M or Isopar N, solvents with equivalent or identical properties but from other suppliers, wherein said solvents are preferably not being consumed or evaporated during the fermentation process, and/or wherein the color of the produced retinyl acetate is transparent.
  • the host cell to be used for production of a retinoid-mix according to the present invention comprises further modifications, such as modification in endogenous enzyme activities leading to conversion of retinol into fatty acid retinyl esters (FAREs).
  • FAREs fatty acid retinyl esters
  • modifications include reduction or deletion of endogenous lipase activities as dependent on the corresponding host cell, particularly reduction or deletion of activity of one or more endogenous gene(s) encoding enzymes with activity equivalent to Yarrowia LIP2 and/or LIP3 and/or LIP4 and/or LIP8, being reduced or abolished, such as polypeptides with at least about 50%, such as 60, 70, 80, 90, 95, 98, or 100% identity to SEQ ID NO:7, 9, 11, 13, or combinations thereof as disclosed in WO2021136689, wherein SEQ ID NO:7 of WO2021136689 corresponds to LIP2 obtainable from Yarrowia lipolytica , SEQ ID NO:9 of WO2021136689 corresponds to LIP3 obtainable from Yarrowia lipolytica , SEQ ID NO:11 of WO2021136689 corresponds to LIP4 obtainable from Yarrowia lipolytica , SEQ ID NO:13 of WO2021136689 correspond
  • the process as defined herein comprising an RDH22 homolog as described herein is modified in the activity of a lipase corresponding to activity of Yarrowia LIP8, such as particularly with reduced or abolished activity, more particularly abolished LIP8 activity, including reduced or abolished activity of a gene encoding a lipase with activity corresponding to LIP8 activity from Yarrowia lipolytica , more preferably wherein a polypeptide with at least about 50% identity to SEQ ID NO:13 of WO2021136689 is abolished.
  • a process for reduction of FARE using a host cell with reduced lipase activity is known from e.g. WO2021136689.
  • lipase is used interchangeably herein with the term “enzyme having lipase activity”. It refers to enzymes involved in pre-digestion of triglyceride oils such as e.g. vegetable oil into glycerol and fatty acids that are normally expressed in oleaginous host cells. Suitable enzymes to be modified in a host cell as defined herein might be selected from endogenous enzymes belonging to EC class 3.1.1.-, including, but not limited to one or more enzyme(s) with activities corresponding to Yarrowia LIP2, LIP3, LIP4, or LIP8 activities.
  • an enzyme having “reduced or abolished” activity means a decrease in its specific activity, i.e. reduced/abolished ability to catalyze formation of a product from a given substrate.
  • a reduction by 100% is referred herein as abolishment of enzyme activity, achievable e.g. via deletion, insertions, frameshift mutations, missense mutations or premature stop-codons in the endogenous gene encoding said enzyme or blocking of the expression and/or activity of said endogenous gene(s) with known methods.
  • sequence identity in order to determine the percentage of sequence identity of two amino acid sequences or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes. In order to optimize the alignment between the two sequences gaps may be introduced in any of the two sequences that are compared. Such alignment can be carried out over the full length of the sequences being compared. Alternatively, the alignment may be carried out over a shorter length, for example over about 20, about 50, about 100 or more nucleic acids/bases or amino acids.
  • sequence identity is the percentage of identical matches between the two sequences over the reported aligned region.
  • the percent 20) sequence identity between two amino acid sequences or between two nucleotide sequences may be determined using the Needleman and Wunsch algorithm for the alignment of two sequences (Needleman, S. B. and Wunsch, C. D. (1970) J. Mol. Biol. 48, 443-453). Both amino acid sequences and nucleotide sequences can be aligned by the algorithm.
  • the Needleman-Wunsch algorithm has been implemented in the computer program NEEDLE.
  • the NEEDLE program from the EMBOSS package was used (version 2.8.0 or higher, EMBOSS: The European Molecular Biology Open Software Suite (2000) Rice, Longden and Bleasby, Trends in Genetics 16, (6) pp 276-277, http://emboss.bioinformatics.nl/).
  • EMBOSS European Molecular Biology Open Software Suite (2000) Rice, Longden and Bleasby, Trends in Genetics 16, (6) pp 276-277, http://emboss.bioinformatics.nl/).
  • EBLOSUM62 is used for the substitution matrix.
  • EDNAFULL is used for nucleotide sequence.
  • the optional parameters used are a gap-open penalty of 10 and a gap extension penalty of 0.5. The skilled person will appreciate that all these different parameters will yield slightly different results but that the overall percentage identity of two sequences is not significantly altered when using different algorithms.
  • the percentage of sequence identity between a query sequence and a sequence of the invention is calculated as follows: number of corresponding positions in the alignment showing an identical amino acid or identical nucleotide in both sequences divided by the total length of the alignment after subtraction of the total number of gaps in the alignment.
  • the identity as defined herein can be obtained from NEEDLE by using the NOBRIEF option and is labeled in the output of the program as “longest identity”. If both amino acid sequences which are compared do not differ in any of their amino acids, they are identical or have 100% identity.
  • the enzymes as described herein to be expressed in a suitable host cell to be used in the present invention also encompass enzymes carrying (further) amino acid substitution(s) which do not alter enzyme activity, i.e. which show the same properties with respect to the enzymes defined herein. Such mutations are also called “silent mutations”. Examples of silent mutations included in the present invention are host-optimized sequences, including but not limited to SEQ ID NO: 3 or 6.
  • the present invention is related to production of a retinoid-mix as defined herein, comprising a percentage of dihydroretinoids of less than about 10% based on total retinoids, comprising a percentage of at least about 80% retinyl acetate based on total retinoids, comprising a percentage of less than about 8% retinal based on total retinoids, wherein the process is conducted in Yarrowia lipolytica , particularly wherein endogenous lipase activities are reduced or completely abolished, such as e.g.
  • LIP8 furthermore comprising reduction or complete abolishment of lipase activities including endogenous LIP2, and/or LIP3, and/or LIP4, said host cell furthermore expressing a heterologous ATF1 homolog, preferably fungal ATF1 with activity of LmATF1, LfATF1, LffATF1, SbATF1 as e.g.
  • WO2019058001 or a modified ATF1 comprising amino acid substitutions S480Q_G409A_V4071_H69A_1484L such as obtainable from Lachancea mirantina , including an enzyme with an identity of at least about 20% to the LmATF1 according to SEQ ID NO:1 as disclosed in WO2020141168, and wherein said host cell comprises and expresses an RDH22 homolog as defined herein, such as e.g. RDH22 with at least about 30% identity to SEQ ID NO:1 or 4.
  • organisms such as e.g. microorganisms, fungi, algae or plants also include synonyms or basonyms of such species having the same physiological properties, as defined by the International Code of Nomenclature of Prokaryotes or the International Code of Nomenclature for algae, fungi, and plants (Melbourne Code).
  • strain Lachancea mirantina is a synonym of strain Zygosaccharomyces sp. IFO 11066, originated from Japan.
  • the term “specific activity” or “activity” with regards to enzymes means its catalytic activity, i.e. its ability to catalyze formation of a product from a given substrate.
  • the specific activity defines the amount of substrate consumed and/or product produced in a given time period and per defined amount of protein at a defined temperature.
  • specific activity is expressed in ⁇ mol substrate consumed or product formed per min per mg of protein.
  • An enzyme is active, if it performs its catalytic activity in vivo, i.e. within the host cell as defined herein or within a suitable (cell-free) system in the presence of a suitable substrate.
  • the skilled person knows how to measure enzyme activity.
  • Analytical methods to evaluate the capability/enzymatic activity of a suitable enzyme involved in retinoid production are known in the art, such as e.g. described in Example 4 of WO2014096992.
  • titers of products such as dihydroretinoids, retinyl acetate, retinol, retinal, beta-carotene, and the like can be measured by HPLC.
  • an enzyme is “expressed and active in vivo” if mRNA encoding for the protein can be detected by Northern blotting and/or protein is detected by mass spectrometry.
  • lipase activity means ability of the host cell to utilize triglycerides according to the definition herein.
  • ATFs as defined herein it means ability of a host cell for acetylation of retinol into retinyl acetate.
  • RDH activity as defined herein it means ability of a host cell for conversion of retinal into retinol.
  • Retinoids as used herein include beta-carotene cleavage products also known as apocarotenoids, including but not limited to retinal, retinolic acid, retinol, retinoic methoxide, retinyl acetate, retinyl esters, 4-keto-retinoids, 3 hydroxy-retinoids, dihydroretinoids or combinations thereof.
  • a mixture comprising one or more of said beta-carotene cleavage products, particularly retinal, retinol, retinyl acetate, and/or dihydroretinoid is defined herein as “retinoid-mix” or “total retinoids”, wherein said retinoids are accumulated during the fermentation process with typical measurement at the end of fermentation process. Biosynthesis of retinoids is described in e.g. WO2008042338.
  • a host cell capable of production of retinoids/retinoid-mix as defined herein in e.g. a fermentation process is known as “retinoid-producing host cell” and includes a host cell producing e.g. retinal, retinol and/or retinyl acetate.
  • retinoid-producing host cell includes a host cell producing e.g. retinal, retinol and/or retinyl acetate.
  • a “retinal-producing host cell” is a host cell wherein the respective polypeptides are expressed and active in vivo, leading to production of retinal, e.g. via enzymatic conversion of beta-carotene into retinal.
  • a “retinol-producing host cell” is a host cell, wherein the respective polypeptides are expressed and active in vivo, leading to production of retinol, e.g. via enzymatic conversion of retinal into retinol.
  • a “retinyl acetate-producing host cell” is the respective host cell capable of acetylation of retinol into retinyl acetate via expression of the respective acetylating enzymes, e.g. ATFs, preferably ATF1 enzymes, as described herein.
  • Retinal as used herein is known under IUPAC name (2E,4E,6E,8E)-3,7-Dimethyl-9-(2,6,6-trimethylcyclohexen-1-yl) nona-2,4,6,8-tetraenal. It is herein interchangeably referred to as retinaldehyde or vitamin A aldehyde and includes both cis- and trans-isomers, such as e.g. 11-cis retinal, 13-cis retinal, trans-retinal and all-trans retinal.
  • carotenoids as used herein is well known in the art. It includes long, 40 carbon conjugated isoprenoid polyenes that are formed in nature by the ligation of two 20 carbon geranylgeranyl pyrophosphate molecules. These include but are not limited to phytoene, lycopene, and carotene, such as e.g. beta-carotene, which can be oxidized on the 4-keto position or 3-hydroxy position to yield canthaxanthin, zeaxanthin, or astaxanthin. Biosynthesis of carotenoids is described in e.g. WO2006102342.
  • Vitamin A as used herein may be any chemical form of vitamin A found in aqueous solutions, in solids and formulations, and includes retinol, retinyl acetate and retinyl esters. It also includes retinoic acid, such as for instance undissociated, in its free acid form or dissociated as an anion.
  • FAREs or “retinyl fatty esters” as used interchangeably herein includes long chain retinyl esters. These long chain retinyl esters define hydrocarbon esters that consists of at least about 8, such as e.g. 9, 10, 12, 13, 15 or 20 carbon atoms and up to about 26, such as e.g. 25, 22, 21 or less carbon atoms, with preferably up to about 6 unsaturated bonds, such as e.g. 0, 1, 2, 4, 5, 6 unsaturated bonds.
  • Long chain retinyl esters include but are not limited to linoleic acid, oleic acid, or palmitic acid.
  • the present invention features the following embodiments 1 to 14:
  • Shake plate assay typically, 200 ⁇ l of 0.075% Yeast extract, 0.25% peptone (0.25X YP) is inoculated with 10 ⁇ l of freshly grown Yarrowia and overlaid with 200 ⁇ l of Isopar M with 2% oleic acid as a carbon source.
  • Clonal isolates of transformants were grown in 24 well plates (Multitron, 30° C., 800 RPM) in YPD media with Isopar M overlay indicated earlier for 4 days. The overlay fraction was removed from the shake plate wells and analyzed by HPLC on a normal phase column, with a photo-diode array detector.
  • DNA transformation Strains were transformed by overnight growth on YPD plate media; 50 ⁇ l of cells were scraped from a plate and transformed by incubation in 500 ⁇ l with 1 ⁇ g transforming DNA, typically linear DNA for integrative transformation, 40% PEG 3550MW, 100 mM lithium acetate, 50 mM Dithiothreitol, 5 mM Tris-Cl pH 8.0, 0.5 mM EDTA for 60 minutes at 40° C. and plated directly to selective media or in the case of dominant antibiotic marker selection the cells were out grown on YPD liquid media for 4 hours at 30° C. before plating on the selective media.
  • transforming DNA typically linear DNA for integrative transformation, 40% PEG 3550MW, 100 mM lithium acetate, 50 mM Dithiothreitol, 5 mM Tris-Cl pH 8.0, 0.5 mM EDTA for 60 minutes at 40° C.
  • plated directly to selective media or in the case of dominant antibiotic marker selection the cells were out grown on
  • Nourseothricin (Nat) selection was performed on YPD media containing 100 ⁇ g/mL nourseothricin and hygromycin (Hyg) selection was performed on YPD containing 100 ⁇ g/mL hygromycin.
  • URA3 marker recycling was performed using 5-fluoroorotic acid (FOA).
  • Episomal hygromycin resistance marker (Hyg) plasmids were cured by passage on non-selective media, with identification of Hyg-sensitive colonies by replica plating colonies from non-selective media to hygromycin containing media (100 ⁇ g/mL).
  • Plasmid MB9523 containing expression systems for DrBCO, LmATF-S480Q_G409A_V4071_H69A_1484L, and FfRDH (SEQ ID NO:8) was synthesized at Genscript (Piscataway, NJ, USA). Plasmid MB9523 contains the ‘URA3’ for marker selection in Yarrowia lipolytica transformations. Genes were synthesized with Nhel and Mlul ends in pUC57 vector.
  • the genes were subcloned to the MB5082 ‘URA3’, MB6157 HygR, and MB8327 NatR vectors for marker selection in Yarrowia lipolytica transformations, as in WO2016172282.
  • Hindlll/Xbal for MB5082-based vectors (all marked with URA3 in Table 1, except MB9523), Pvull (for MB6157—and MB8327-based vectors), or Sfil (for MB9523), respectively purified by gel electrophoresis and Qiagen gel purification column. Clones were verified by sequencing.
  • Plasmid MB8388-LIP8 (SEQ ID NO: 9), containing a Cas9, and guide RNA expression systems to target LIP8, was synthesized at Genscript (Piscataway, NJ, USA).
  • Plasmid list Plasmid, strains, nucleotide and amino acid sequences that were used are listed in Table 1, 2, 6 and the sequence listing. In general, all non-modified sequences referred to herein are the same as the accession sequence in the database for reference strain CLIB122 (Dujon B, et al, Nature. 2004 Jul. 1;430 (6995): 35-44).
  • LmATF1-mut refers to Lachancea mirantina (LmATF1; SEQ ID NO: 13 in WO2019058001) carrying aa substitutions S480Q_G409A_V4071_H69A_1484L.
  • DrBCO refers to BCO originated from Danio rerio (see SEQ ID NO: 16 in WO2020141168);
  • FfRDH′′ refers to RDH originated from Fusarium (see SEQ ID NO: 22 in WO2020141168). For more explanation, see text.
  • Normal phase retinol method A Waters 1525 binary pump attached to a Waters 717 auto sampler were used to inject samples. A Phenomenex Luna 3u Silica (2), 150 ⁇ 4.6 mm with a security silica guard column kit was used to resolve retinoids.
  • the mobile phase consists of either, 1000 mL hexane, 30 mL isopropanol, and 0.1 mL acetic acid for astaxanthin related compounds, or 1000 mL hexane, 60 mL isopropanol, and 0.1 mL acetic acid for zeaxanthin related compounds. The flow rate for each was 0.6 mL per minute. Column temperature was ambient. The injection volume was 20 ⁇ L. The detector was a photodiode array detector collecting from 210 to 600 nm. Analytes were detected according to Table 3.
  • RT means retention time.
  • Sample preparation Samples were prepared by various methods depending on the conditions. For whole broth or washed broth samples the broth was placed in a Precellys® tube weighed and mobile phase was added, the samples were processed in a Precellys® homogenizer (Bertin Corp, Rockville, MD, USA) on the highest setting 3X according to the manufactures directions. In the washed broth the samples were spun in a 1.7 ml tube in a microfuge at 10000 rpm for 1 minute, the broth decanted, 1 ml water added mixed pelleted and decanted and brought up to the original volume the mixture was pelleted again and brought up in appropriate amount of mobile phase and processed by Precellys® bead beating.
  • the sample was spun at 4000 RPM for 10 minutes and the oil was decanted off the top by positive displacement pipet (Eppendorf, Hauppauge, NY, USA) and diluted into mobile phase mixed by vortexing and measured for retinoid concentration by HPLC analysis.
  • positive displacement pipet Eppendorf, Hauppauge, NY, USA
  • Fermentation conditions Fermentations were identical to the previously described conditions using Isopar M overlay and stirred tank that was ethanol fed in a bench top reactor with 0.5L to 5L total volume (see WO2016172282). Generally, the same results were observed with a fed batch stirred tank reactor with an increased productivity demonstrating the utility of the system for the production of retinoids.
  • % total retinoids is the percentage of retinoids produced compared to total retinoids in the control without addition of plasmids, wherein the total retinoids obtained with the control is set to 100%.
  • % retinal means percentage of retinal in the retinoid mix based on total retinoids. For more explanation, see text.
  • HsRDH12 MB8203
  • WaRDH22 MB9997
  • YlRDH22 ML18812-lip8
  • HsRDH12 and YlRDH22 MB9894
  • addition of either HsRDH12, WaRDH22, YlRDH22, or both HsRDH12 and YlRDH22 increases retinyl acetate purity.
  • nucleotide sequences with a ′′*′′ refer to codon-optimized sequences.
  • ′′aa′′ refers to amino acids sequence,
  • ′′nt′′ refers to nucleotide sequence.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Genetics & Genomics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Mycology (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Plant Pathology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

The present invention is related to bio-production of retinoids with improved purity profile, particularly bio-retinoids with a high percentage of retinyl acetate, wherein the flux towards retinol is increased and wherein the percentage of unwanted by-products such as dihydroretinoids or retinal is minimized to a range of below 10% by expressing retinol dehydrogenase (EC 1.1.1.105, RDH22) derived from Yarrowia lipolytica (Candida lipolytica) or Wickerhamomyces anomalus (Hanensula anomala).

Description

  • The present invention is related to bio-production of retinoids with improved purity profile, particularly bio-retinoids with a high percentage of retinyl acetate, wherein the flux towards retinol is increased and wherein the percentage of unwanted by-products such as dihydroretinoids is minimized to a range of below 10%.
  • Retinol is an important intermediate/precursor in the process of retinoid production, particularly such as vitamin A production. Retinoids, including vitamin A, are one of very important and indispensable nutrient factors for human beings which have to be supplied via nutrition. Retinoids promote well-being of humans, inter alia in respect of vision, the immune system and growth.
  • Current chemical production methods for retinoids, including vitamin A and precursors thereof, have some undesirable characteristics such as e.g. high-energy consumption, complicated purification steps and/or undesirable by-products. Therefore, over the past decades, other approaches to manufacture retinoids, including vitamin A and precursors thereof, including microbial conversion steps, which would be more economical as well as ecological, have been investigated.
  • Unfortunately, the biological systems that produce retinoids are industrially intractable and/or produce the compounds at such low levels that commercial scale isolation is not practical. The most limiting factors include instability of intermediates including instability of retinol, inefficient enzymatic conversions and/or impurity due to accumulation of unwanted side-products.
  • Thus, it is an ongoing task to look for an improved process for fermentative production of retinoids, particularly with a high content of stable forms such as e.g. retinyl acetate, and at the same time to reduce the formation of unwanted by-products such as e.g. dihydroretinoids.
  • Surprisingly, we now identified a novel class of retinal reducing enzymes (RDHs) that are involved in formation of retinol, wherein the use of said enzymes in a fermentative production of retinoids leads to about 90% reduction of by-products such as dihydroretinoids and retinal that are generated during said fermentation process.
  • Thus, the present invention is related to a process for production of a retinoid-mix, such as a fermentation process using a suitable retinoid-producing host cell, said retinoid-mix comprising dihydroretinoids with a percentage of less than about 10% based on total retinoids within said mix.
  • As used herein, a “retinoid mix” is defined as all retinoids formed during a retinoid production process, such as e.g. fermentation process using a suitable host cell that is expressing suitable enzymes involved in formation of retinoids including but not limited to retinal, retinol, and retinyl acetate and optionally furthermore expressing enzymes involved in formation of beta-carotene. Said retinoid mix comprises retinal, retinol, retinyl acetate, and dihydroretinods.
  • Particularly, such retinoid-mix according to the present invention comprises dihydroretinoids, retinal, and retinyl acetate, wherein the percentage of dihydroretinoids is less than about 10% based on total retinoids, preferably wherein the percentage of retinal is less than about 8% based on total retinoids, further preferably wherein the percentage of retinyl acetate is at least about 80% based on total retinoids, even further preferably wherein the percentage of trans-isomers within said mix is at least about 80% based on the amount of all cis- and trans-retinoids.
  • In one embodiment, the percentage of dihydroretinoids based on total retinoids within said retinoid-mix is about less than 10, such as 9, 8, 7, 6, 5, 4, 3, 1% or less, preferably 2% or less, more preferably 1% or less, such as 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1% or less, such as in the range of 1% to 0.1, 0.01, or 0.001%, as measurable at the end of the production or fermentation process.
  • In one embodiment, the percentage of retinal based on total retinoids within said retinoid-mix is about less than 8, such as 7, 6, 5, 4, 3, 1% or less, preferably 2% or less, more preferably 1% or less, such as 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1% or less, such as in the range of 1% to 0.1, 0.01, or 0.001%, as measurable at the end of the production or fermentation process.
  • In a further embodiment, the percentage of retinyl acetate based on total retinoids within said retinoid-mix is at least about 80%, such as e.g. 85, 90, 92, 95, 98% or more retinyl acetate, as measurable at the end of the production or fermentation process using a suitable retinyl-acetate producing host cell.
  • In another embodiment, the percentage of trans-isoforms based on the total amount of cis and trans-isomers in said retinoid-mix is at least about 80, 85, 90, 92, 95, 98% or more, as measurable at the end of the production or fermentation process. Particularly, said mix comprises at least about 80% of trans-retinyl acetate, i.e. 80% of the retinyl acetate in the retinoid-mix is present in the trans-isomer.
  • The present invention is related to a biotechnological process for production of a retinoid-mix as defined herein comprising enzymatic conversion of retinal into retinol in the presence of an RDH22 homolog, particularly obtainable from fungi, as defined herein, particularly comprising cultivation of a suitable host cell, more particularly a retinal-producing host cell, expressing said RDH22 homolog under suitable conditions that allow formation of said retinoid-mix as defined herein.
  • The terms “RDH”, “retinal reductase”, “retinal reducing enzyme”, “enzyme involved in formation of retinol” or “enzyme involved in conversion of retinal into retinol” are used interchangeably herein and refer to enzymes [EC 1.1.1.105] which are involved in the conversion/catalysis of retinal into retinol as well as the back-conversion from retinol to retinal. With regards to RDH22, the formation of dihydroretinoids is reduced to a percentage of less than about 10% detectable in a retinoid-mix formed during a fermentation process using a suitable retinoid-producing host cell and under suitable conditions and the flux towards retinol is increased such that the percentage of retinal present in said retinoid-mix as detectable at the end of the fermentation process is less than about 8% based on total retinoids.
  • Thus, the present invention is related to RDH22 homologs as defined herein capable of catalyzing retinal into retinol, wherein the conversion towards retinol is increased, leading to a retinoid-mix with a percentage of less than about 8% retinal based on total retinoids, which can be measured at the end of the fermentation process by known methods, particularly process for production of a retinoid-mix with at least about 80% retinyl acetate as described herein.
  • In one embodiment, the present invention is directed to RDH22 homologs obtainable from Yarrowia, preferably Yarrowia lipolytica, comprising YlRDH22 according to SEQ ID NO:1 and enzymes originated from other source organisms or being artificially constructed (based on digital sequence information) but having activity corresponding to the respective Yarrowia RDH22 according to SEQ ID NO: 1. Using such YlRDH22 homolog as defined herein in a process comprising production of retinol and retinyl acetate, the percentage of retinal accumulated during such production process and present in the retinoid-mix measured at the end of the production process could be reduced to less than 1% based on total retinoids.
  • Preferably, the present invention is directed to polypeptides with at least about 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 92, 95, 97, 98, 99% or up to 100% identity to SEQ ID NO:1 including polynucleotides encoding said polypeptides, such as e.g. polynucleotide according to SEQ ID NO:2 or 3 as well as enzymes having equivalent enzymatic activity, i.e. activity corresponding to YlRDH22 as defined above but are originated from another source organism or being based on digital sequence information, particularly being originated from another fungal organism.
  • In one embodiment, the present invention is directed to RDH22 homologs obtainable from Wickerhamomyces, preferably Wickerhamomyces anomalus, comprising WaRDH22 according to SEQ ID NO:4 and enzymes originated from other source organisms or being artificially constructed (based on digital sequence information) but having activity corresponding to the respective Wickerhamomyces RDH22 according to SEQ ID NO:4. Using such WaRDH22 homolog as defined herein in a process comprising production of retinol and retinyl acetate, the percentage of retinal accumulated during such production process and present in the retinoid-mix measured at the end of the production process could be reduced to about 2% or less based on total retinoids. with a conversion rate of more than 90% towards formation of retinol.
  • Preferably, the present invention is directed to polypeptides with at least about 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 92, 95, 97, 98, 99% or up to 100% identity to SEQ ID NO:4 including polynucleotides encoding said polypeptides, such as e.g. polynucleotide according to SEQ ID NO:5 or 6 as well as enzymes having equivalent enzymatic activity, i.e. activity corresponding to WaRDH22 as defined above but are originated from another source organism or being based on digital sequence information, particularly being originated from another fungal organism.
  • The RDH22 enzymes as defined herein are catalyzing the conversion of retinal into retinol with a conversion being shifted towards production of retinol, wherein the substrate (i.e. retinal) can be either cis-, trans- or a mix of cis-/trans-retinal in any possible ratio, with a percentage of at least about 80% trans-isomers being preferred. Conversion of said retinal-mix with about 80% trans-retinal would lead to retinol and optionally retinyl acetate with about the same ratio of trans to cis-isomers in a respective retinoid-mix obtained by a process for fermentative production as defined herein, such as starting from beta-carotene using a host cell capable of retinal, retinol and/or retinyl-acetate production. The skilled person knows how to generate retinal from beta-carotene using beta-carotene oxygenases (BCOs), such as exemplified in WO2019057999 or WO2021009194. Enzymes, particularly acetyl transferases (ATFs), more preferably ATF1s, host cells and processes for conversion of retinol into retinyl acetate are known, such as e.g. from WO2019058000 or WO2020141168.
  • The terms “conversion” “enzymatic conversion or “cleavage” in connection with enzymatic catalysis of substrates such as e.g. beta-carotene, retinal or retinol are used interchangeably herein and refer to the action of the specific enzymes, including but not limited to BCOs, RDHs, particularly RDH22, or ATFs as defined herein involved in formation of retinal, retinol or retinyl acetate.
  • As used herein, the term “dihydroretinoids” and “13,14-dihydroretinoids” are used interchangeably herein and includes but not limited to dihydroretinol, dihydroretinal, dihydroretinyl acetate that can be detected in a retinoid-mix according to the present invention. The skilled person known how to measure this.
  • Particularly, the present invention is related to a process for reducing the percentage of by-products including e.g. retinal and dihydroretinoids present in a retinoid-mix as defined herein, particularly wherein a retinoid-producing host cell, such as a retinal/retinol-producing host cell is modified leading to (over) expression of enzymes having RDH22 activity, particularly YlRDH22 or WaRDH22 and homologs thereof as defined herein, wherein the reduction of retinal is in the range of 20 to more than 92%, particularly about 25, 30, 40, 50, 70, 75, 80, 85, 90, 92, 95% or more and/or the reduction of dihydroretinoids is in the range of 20 to more than 97%, particularly about 25, 30, 40, 50, 70, 75, 80, 85, 90, 92, 95, 97% or more as compared to a process using a non-modified host cell, i.e. a host cell that has not been transformed with such heterologous RDH22 polynucleotides as defined herein.
  • As used herein, a “modified host cell” refers to a host cell or a process wherein the host cell is (over) expressing a heterologous RDH22 homolog as defined herein, including but not limited to host cells modified such that the activity of an endogenous RDH22 homolog as defined herein is increased as compared to the wild-type or non-modified host cell, such as e.g. via expression of 2, 3, 4, or more gene copies of an endogenous gene, i.e. expression of more copies of the respective endogenous gene as occurring in the respective non-modified wild-type host cell. A modified host cell is also known as “recombinant host cell”.
  • As used herein, a “non-modified host cell” or “wild-type host cell”-which is used interchangeably herein-refers to the respective host cell that is not transformed with a heterologous RDH22 homolog as defined herein and/or that is not over-expressing an endogenous RDH22 homolog, such as e.g. an enzyme having YlRDH22 or WaRDH22 activity as defined herein.
  • Modifications in order to generate a recombinant host cell having increased activity of an RDH22 homolog as defined herein, may include the use of strong promoters, suitable transcriptional- and/or translational enhancers, or the introduction of one or more gene copies into the respective wild-type host cell, leading to increased accumulation of the respective enzymes and the respective products (e.g. retinol, retinyl acetate) in a given time. The skilled person knows which techniques to use in dependence of the host cell. The increase (or reduction) of gene expression can be measured by various methods, such as e.g. Northern, Southern, or Western blot technology as known in the art.
  • Expression of the novel RDH22 homologs as defined herein can be achieved in any host system, including (micro) organisms, which is suitable for conversion of retinal into retinol (i.e. retinol-producing host cell) and further suitable for conversion of retinol into retinyl acetate (i.e. retinyl-acetate producing host cell) and furthermore suitable for conversion of carotenoid precursors into beta-carotene (i.e. carotenoid-producing host cell) and furthermore suitable for conversion of beta-carotene into retinal (i.e. retinal-producing host cell) and which allows the expression of the enzymes as disclosed herein and as used for such processes. Examples of suitable host cells to be used for the present invention might be selected from bacteria, algae, fungi including yeast, plant or animal cells, such as e.g. fungal host cells including oleaginous yeast cells, such as e.g. Rhodosporidium, Lipomyces, Saccharomyces or Yarrowia, preferably Yarrowia, more preferably Yarrowia lipolytica or bacterial host cells including but not limited to Escherichia or Pantoea. The skilled person knows which genes are suitable in order to express the necessary enzymes involved in carotenoid—and/or retinoid biosynthesis in a suitable host cell. Genes and methods to generate carotenoid-producing host cells are known in the art, see e.g. WO2006102342. Depending on the carotenoid to be produced, different genes might be involved.
  • As used herein, the term “fungal host cell” particularly includes yeast cells, such as e.g. retinal/retinol/retinyl-acetate-producing yeast cells, comprising Yarrowia or Saccharomyces. As used herein, the term retinyl-acetate producing host cells” includes host cells, capable of synthesizing retinol and expressing acetyl transferases (ATFs) as defined in e.g. WO2019058001 or WO2020141168 resulting in retinyl acetate with a percentage as defined herein based on total retinoids produced by said host cell. Optionally, such host cell is furthermore capable of producing carotenoids.
  • Preferably, the host cell expressing an enzyme having activity of YlRDH22 or WaRDH22 as defined herein, including enzymes with at least about 30% identity to YlRDH22 according to SEQ ID NO:1 or WaRDH22 according to SEQ ID NO:4 to be used for fermentative production of a retinoid-mix according to the present invention comprises further modifications enabling the conversion of retinol into retinyl acetate via action/expression of heterologous ATF [EC 2.3.1.84], 20) particularly fungal ATF, comprising a highly conserved partial amino acid sequence of at least 7 amino acid residues selected from [NDEHCS]-H-x(3)-D-[GA] (motifs are in Prosite syntax, as defined in https://prosite.expasy.org/scanprosite/scanprosite_doc.html), wherein “x” denotes an arbitrary amino acid and with the central histidine being part of the enzyme's binding pocket, preferably wherein the 7 amino acid motif is selected from [NDE]-H-x(3)-D-[GA], more preferably selected from [ND]-H-x (3)-D-[GA], most preferably selected from N—H-x (3)-D-[GA] corresponding to position N218 to G224 in the polypeptide according to SEQ ID NO:1 as disclosed in WO2020141168. Examples of such enzymes might be particularly selected from L. mirantina, L. fermentati, S. bayanus, or W. anomalus, such as disclosed in WO2020141168 or WO2019058001, more preferably said ATFs comprising one or more amino acid substitution(s) in a sequence with at least about 20%, such as e.g. 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 92, 95, 97, 98, 99% or up to 100% identity to LmATF1 (i.e. SEQ ID NO:1 as disclosed in WO2020141168), wherein the one or more amino acid substitution(s) are located at position(s) corresponding to amino acid residue(s) selected from the group consisting of position 68, 69, 72, 73, 171, 174, 176, 178, 291, 292, 294, 301, 307, 308, 311, 312, 320, 322, 334, 362, 405, 407, 409, 480, 483, 484, 490, 492, 520, 521, 522, 524, 525, 526 and combinations thereof and as particularly exemplified in Table 4 of WO2020141168, most preferably comprising one or more amino acid substitution(s) on positions corresponding to amino acid residue(s) 69, 407, 409, 480, 484, and combinations thereof in SEQ ID NO:1 as disclosed in WO2020141168, even more preferably comprising an amino acid substitution at a position corresponding to residue 69 in the polypeptide according to SEQ ID NO: 1 as disclosed in WO2020141168 leading to asparagine, serine or alanine at said residue, such as e.g. via substitution of histidine by asparagine (H69N), serine (H69S) or alanine (H69A), with preference for H69A, optionally being combined with amino acid substitution at a position corresponding to residue 407 in the polypeptide according to SEQ ID NO:1 as disclosed in WO2020141168 leading to isoleucine at said residue, such as e.g. via substitution of valine by isoleucine (V4071), optionally being combined with an amino acid substitution at a position corresponding to residue 409 in the polypeptide according to SEQ ID NO: 1 as disclosed in WO2020141168 leading to alanine at said residue, such as e.g. via substitution of glycine by alanine (G409A), optionally being combined with amino acid substitution at a position corresponding to residue 480 in the polypeptide according to SEQ ID NO:1 as disclosed in WO2020141168 leading to glutamic acid, lysine, methionine, phenylalanine or glutamine at said residue, such as e.g. via substitution of serine by glutamic acid (S480E), lysine (S480L), methionine (S480M), phenylalanine (S480F) or glutamine (S480Q), optionally being combined with amino acid substitution at a position corresponding to residue 484 in the polypeptide according to SEQ ID NO:1 as disclosed in WO2020141168 leading to leucine at said residue, such as e.g. via substitution of isoleucine by leucine (1484L). Said modified enzyme might be originated from yeast, such as e.g. L. mirantina, L. fermentati, W. anomalus or S. bayanus, preferably from L. mirantina. In a most preferred embodiment, the ATF to be used for the process according to the present invention is a modified ATF1 comprising amino acid substitutions S480Q_G409A_V4071_H69A_1484L and is obtainable from Lachancea mirantina, including an enzyme with an identity of at least about 20% to the LmATF1 according to SEQ ID NO:1 as disclosed in WO2020141168.
  • Suitable culture conditions of a fermentation process according to the present invention include cultivation of the host cell in an aqueous medium in the presence of suitable carbon sources as defined herein, optionally supplemented with appropriate nutrients under aerobic or anaerobic conditions and as known by the skilled person to enable production of a retinoid-mix as defined herein. The fermentation may be conducted in batch, fed-batch, semi-continuous or continuous mode. Particularly, fermentations are run in fed-batch stirred tank reactors. Fermentations can be run for 5 to 14 days, such as e.g. for around 118 h. Fermentation products including but not limited to retinol, retinyl acetate may be harvested from the cultivation at a suitable moment, e.g. when the tank fills due to addition of the feed. The retinoid-mix comprising retinol and acetylated retinoids might be further purified and/or further processed/formulated, to be used in the food, feed, pharma or cosmetic industry. Cultivation and isolation of host cells selected from Yarrowia or Saccharomyces to be used for production of carotenoids and/or retinoids is described in e.g. WO2008042338.
  • Carbon sources to be used for the present invention might be selected from linear alkanes, free fatty acids, including triglycerides, particularly vegetable oil, such as e.g. selected from the group consisting of oil originated from corn, soy, olive, sunflower, canola, cottonseed, rapeseed, sesame, safflower, grapeseed or mixtures thereof, including the respective free fatty acids, such as e.g. oleic acid, palmitic acid or linoleic acid. Suitable carbon sources might furthermore be selected from ethanol, glycerol or glucose and mixtures of one or more of the above-listed carbon sources.
  • Particularly, the present invention is directed to a process for the production of retinoids, in particular a retinoid-mix as defined herein, in a two-phase culture system including an in vitro extraction system, i.e. cultivation in the presence of a lipophilic solvent as defined herein, wherein a retinal-/retinol-/retinyl acetate-producing host cell, preferably oleaginous yeast cell such as e.g. Yarrowia, is cultivated under suitable culture conditions such that the retinoid-mix, including retinyl acetate, is accumulated in the lipophilic solvent and optionally extracted/isolated and/or purified from said lipophilic solvent.
  • Preferred lipophilic solvents to be used for this aspect of the present invention might be selected from isoparaffins including mixtures of alkanes, cycloparaffin, isoalkanes, cycloalkanes, or dodecanes. The solvents might be natural or synthetic ones. Examples of commercially available useful solvents might be selected from Total, e.g. Isane® solvents, Shell, e.g. ShellSolTD or ShellSolT, Exxon Mobile, e.g. Isopar™ fluids, particularly such as e.g. Isopar M, Isopar N, Isopar H, Isopar K, Isopar L, or mixtures with iso-dodecane isomers, as e.g. commercially available under the tradename AC365770010 (Acros Organics).
  • Preferably, the second phase solvent is selected from isoparaffins, such as e.g. Isopar M, Isopar N, Isopar H, Isopar K, Isopar L, more preferably selected from Isopar L, Isopar M or Isopar N, solvents with equivalent or identical properties but from other suppliers, wherein said solvents are preferably not being consumed or evaporated during the fermentation process, and/or wherein the color of the produced retinyl acetate is transparent.
  • In one particular embodiment, the host cell to be used for production of a retinoid-mix according to the present invention comprises further modifications, such as modification in endogenous enzyme activities leading to conversion of retinol into fatty acid retinyl esters (FAREs). Particularly, such modifications include reduction or deletion of endogenous lipase activities as dependent on the corresponding host cell, particularly reduction or deletion of activity of one or more endogenous gene(s) encoding enzymes with activity equivalent to Yarrowia LIP2 and/or LIP3 and/or LIP4 and/or LIP8, being reduced or abolished, such as polypeptides with at least about 50%, such as 60, 70, 80, 90, 95, 98, or 100% identity to SEQ ID NO:7, 9, 11, 13, or combinations thereof as disclosed in WO2021136689, wherein SEQ ID NO:7 of WO2021136689 corresponds to LIP2 obtainable from Yarrowia lipolytica, SEQ ID NO:9 of WO2021136689 corresponds to LIP3 obtainable from Yarrowia lipolytica, SEQ ID NO:11 of WO2021136689 corresponds to LIP4 obtainable from Yarrowia lipolytica, SEQ ID NO:13 of WO2021136689 corresponds to LIP8 obtainable from Yarrowia lipolytica. Preferably, the process as defined herein comprising an RDH22 homolog as described herein is modified in the activity of a lipase corresponding to activity of Yarrowia LIP8, such as particularly with reduced or abolished activity, more particularly abolished LIP8 activity, including reduced or abolished activity of a gene encoding a lipase with activity corresponding to LIP8 activity from Yarrowia lipolytica, more preferably wherein a polypeptide with at least about 50% identity to SEQ ID NO:13 of WO2021136689 is abolished. A process for reduction of FARE using a host cell with reduced lipase activity is known from e.g. WO2021136689.
  • The term “lipase” is used interchangeably herein with the term “enzyme having lipase activity”. It refers to enzymes involved in pre-digestion of triglyceride oils such as e.g. vegetable oil into glycerol and fatty acids that are normally expressed in oleaginous host cells. Suitable enzymes to be modified in a host cell as defined herein might be selected from endogenous enzymes belonging to EC class 3.1.1.-, including, but not limited to one or more enzyme(s) with activities corresponding to Yarrowia LIP2, LIP3, LIP4, or LIP8 activities.
  • As used herein, an enzyme having “reduced or abolished” activity means a decrease in its specific activity, i.e. reduced/abolished ability to catalyze formation of a product from a given substrate. A reduction by 100% is referred herein as abolishment of enzyme activity, achievable e.g. via deletion, insertions, frameshift mutations, missense mutations or premature stop-codons in the endogenous gene encoding said enzyme or blocking of the expression and/or activity of said endogenous gene(s) with known methods.
  • The terms “sequence identity”, “% identity” are used interchangeable herein. For the purpose of this invention, it is defined here that in order to determine the percentage of sequence identity of two amino acid sequences or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes. In order to optimize the alignment between the two sequences gaps may be introduced in any of the two sequences that are compared. Such alignment can be carried out over the full length of the sequences being compared. Alternatively, the alignment may be carried out over a shorter length, for example over about 20, about 50, about 100 or more nucleic acids/bases or amino acids. The sequence identity is the percentage of identical matches between the two sequences over the reported aligned region. The percent 20) sequence identity between two amino acid sequences or between two nucleotide sequences may be determined using the Needleman and Wunsch algorithm for the alignment of two sequences (Needleman, S. B. and Wunsch, C. D. (1970) J. Mol. Biol. 48, 443-453). Both amino acid sequences and nucleotide sequences can be aligned by the algorithm. The Needleman-Wunsch algorithm has been implemented in the computer program NEEDLE. For the purpose of this invention the NEEDLE program from the EMBOSS package was used (version 2.8.0 or higher, EMBOSS: The European Molecular Biology Open Software Suite (2000) Rice, Longden and Bleasby, Trends in Genetics 16, (6) pp 276-277, http://emboss.bioinformatics.nl/). For protein sequences EBLOSUM62 is used for the substitution matrix. For nucleotide sequence, EDNAFULL is used. The optional parameters used are a gap-open penalty of 10 and a gap extension penalty of 0.5. The skilled person will appreciate that all these different parameters will yield slightly different results but that the overall percentage identity of two sequences is not significantly altered when using different algorithms.
  • After alignment by the program NEEDLE as described above the percentage of sequence identity between a query sequence and a sequence of the invention is calculated as follows: number of corresponding positions in the alignment showing an identical amino acid or identical nucleotide in both sequences divided by the total length of the alignment after subtraction of the total number of gaps in the alignment. The identity as defined herein can be obtained from NEEDLE by using the NOBRIEF option and is labeled in the output of the program as “longest identity”. If both amino acid sequences which are compared do not differ in any of their amino acids, they are identical or have 100% identity.
  • The enzymes as described herein to be expressed in a suitable host cell to be used in the present invention also encompass enzymes carrying (further) amino acid substitution(s) which do not alter enzyme activity, i.e. which show the same properties with respect to the enzymes defined herein. Such mutations are also called “silent mutations”. Examples of silent mutations included in the present invention are host-optimized sequences, including but not limited to SEQ ID NO: 3 or 6.
  • Thus, in a preferred embodiment the present invention is related to production of a retinoid-mix as defined herein, comprising a percentage of dihydroretinoids of less than about 10% based on total retinoids, comprising a percentage of at least about 80% retinyl acetate based on total retinoids, comprising a percentage of less than about 8% retinal based on total retinoids, wherein the process is conducted in Yarrowia lipolytica, particularly wherein endogenous lipase activities are reduced or completely abolished, such as e.g. activity of LIP8, furthermore comprising reduction or complete abolishment of lipase activities including endogenous LIP2, and/or LIP3, and/or LIP4, said host cell furthermore expressing a heterologous ATF1 homolog, preferably fungal ATF1 with activity of LmATF1, LfATF1, LffATF1, SbATF1 as e.g. disclosed in WO2019058001 or a modified ATF1 comprising amino acid substitutions S480Q_G409A_V4071_H69A_1484L such as obtainable from Lachancea mirantina, including an enzyme with an identity of at least about 20% to the LmATF1 according to SEQ ID NO:1 as disclosed in WO2020141168, and wherein said host cell comprises and expresses an RDH22 homolog as defined herein, such as e.g. RDH22 with at least about 30% identity to SEQ ID NO:1 or 4.
  • With regards to the present invention, it is understood that organisms, such as e.g. microorganisms, fungi, algae or plants also include synonyms or basonyms of such species having the same physiological properties, as defined by the International Code of Nomenclature of Prokaryotes or the International Code of Nomenclature for algae, fungi, and plants (Melbourne Code). Thus, for example, strain Lachancea mirantina is a synonym of strain Zygosaccharomyces sp. IFO 11066, originated from Japan.
  • As used herein, the term “specific activity” or “activity” with regards to enzymes means its catalytic activity, i.e. its ability to catalyze formation of a product from a given substrate. The specific activity defines the amount of substrate consumed and/or product produced in a given time period and per defined amount of protein at a defined temperature. Typically, specific activity is expressed in μmol substrate consumed or product formed per min per mg of protein. Typically, μmol/min is abbreviated by U (=unit). Therefore, the unit definitions for specific activity of μmol/min/(mg of protein) or U/(mg of protein) are used interchangeably throughout this document.
  • An enzyme is active, if it performs its catalytic activity in vivo, i.e. within the host cell as defined herein or within a suitable (cell-free) system in the presence of a suitable substrate. The skilled person knows how to measure enzyme activity. Analytical methods to evaluate the capability/enzymatic activity of a suitable enzyme involved in retinoid production are known in the art, such as e.g. described in Example 4 of WO2014096992. In brief, titers of products such as dihydroretinoids, retinyl acetate, retinol, retinal, beta-carotene, and the like can be measured by HPLC.
  • As used herein, an enzyme is “expressed and active in vivo” if mRNA encoding for the protein can be detected by Northern blotting and/or protein is detected by mass spectrometry. With regards to the lipase activity as defined herein it means ability of the host cell to utilize triglycerides according to the definition herein. With regards to ATFs as defined herein it means ability of a host cell for acetylation of retinol into retinyl acetate. With regards to RDH activity as defined herein it means ability of a host cell for conversion of retinal into retinol.
  • Retinoids as used herein include beta-carotene cleavage products also known as apocarotenoids, including but not limited to retinal, retinolic acid, retinol, retinoic methoxide, retinyl acetate, retinyl esters, 4-keto-retinoids, 3 hydroxy-retinoids, dihydroretinoids or combinations thereof. A mixture comprising one or more of said beta-carotene cleavage products, particularly retinal, retinol, retinyl acetate, and/or dihydroretinoid is defined herein as “retinoid-mix” or “total retinoids”, wherein said retinoids are accumulated during the fermentation process with typical measurement at the end of fermentation process. Biosynthesis of retinoids is described in e.g. WO2008042338.
  • A host cell capable of production of retinoids/retinoid-mix as defined herein in e.g. a fermentation process is known as “retinoid-producing host cell” and includes a host cell producing e.g. retinal, retinol and/or retinyl acetate. As used herein, a “retinal-producing host cell” is a host cell wherein the respective polypeptides are expressed and active in vivo, leading to production of retinal, e.g. via enzymatic conversion of beta-carotene into retinal. As used herein, a “retinol-producing host cell” is a host cell, wherein the respective polypeptides are expressed and active in vivo, leading to production of retinol, e.g. via enzymatic conversion of retinal into retinol. A “retinyl acetate-producing host cell” is the respective host cell capable of acetylation of retinol into retinyl acetate via expression of the respective acetylating enzymes, e.g. ATFs, preferably ATF1 enzymes, as described herein.
  • “Retinal” as used herein is known under IUPAC name (2E,4E,6E,8E)-3,7-Dimethyl-9-(2,6,6-trimethylcyclohexen-1-yl) nona-2,4,6,8-tetraenal. It is herein interchangeably referred to as retinaldehyde or vitamin A aldehyde and includes both cis- and trans-isomers, such as e.g. 11-cis retinal, 13-cis retinal, trans-retinal and all-trans retinal.
  • The term “carotenoids” as used herein is well known in the art. It includes long, 40 carbon conjugated isoprenoid polyenes that are formed in nature by the ligation of two 20 carbon geranylgeranyl pyrophosphate molecules. These include but are not limited to phytoene, lycopene, and carotene, such as e.g. beta-carotene, which can be oxidized on the 4-keto position or 3-hydroxy position to yield canthaxanthin, zeaxanthin, or astaxanthin. Biosynthesis of carotenoids is described in e.g. WO2006102342.
  • “Vitamin A” as used herein may be any chemical form of vitamin A found in aqueous solutions, in solids and formulations, and includes retinol, retinyl acetate and retinyl esters. It also includes retinoic acid, such as for instance undissociated, in its free acid form or dissociated as an anion. A preferred form of vitamin A is retinyl acetate, wherein the terms “retinyl acetate”, “retinol acetate” and “vitamin A acetate” might be used interchangeably (see https://www.cancer.gov/publications/dictionaries/cancer-drug/def/retinyl-acetate?redirect=true).
  • The terms “triglycerides” and “triglyceride oils” are used interchangeably herein. “FAREs” or “retinyl fatty esters” as used interchangeably herein includes long chain retinyl esters. These long chain retinyl esters define hydrocarbon esters that consists of at least about 8, such as e.g. 9, 10, 12, 13, 15 or 20 carbon atoms and up to about 26, such as e.g. 25, 22, 21 or less carbon atoms, with preferably up to about 6 unsaturated bonds, such as e.g. 0, 1, 2, 4, 5, 6 unsaturated bonds. Long chain retinyl esters include but are not limited to linoleic acid, oleic acid, or palmitic acid.
  • Particularly, the present invention features the following embodiments 1 to 14:
      • Embodiment 1: Process for production of a retinoid mix comprising dihydroretinoids, retinal, retinol, and retinyl acetate, wherein the percentage of dihydroretinoids in said mix is less than about 10% or based on total retinoids.
      • Embodiment 2: Process of embodiment 1, wherein the percentage of retinyl acetate in said mix is at least about 80% based on total retinoids.
      • Embodiment 3: Process of embodiment 1 or 2, wherein the percentage of retinyl acetate in said mix is at least about 80% based on total retinoids.
      • Embodiment 4: Process of embodiment 1, 2, or 3, wherein said mix comprises trans and cis-isomers and wherein the percentage of trans-isomers is of at least 80% based on total cis and trans-isomers in said mix.
      • Embodiment 5: Process of embodiment 4, wherein the percentage of trans-retinyl acetate in said mix is at least about 80%.
      • Embodiment 6: Process of embodiment 1, 2, 3, 4, or 5, wherein said mix comprising dihydroretinal, dihydroretinol and dihydroretinyl acetate.
      • Embodiment 7: A retinol-producing host cell expressing a retinol dehydrogenase 22 (RDH22) homolog with at least about 30% identity to a polynucleotide according to SEQ ID NO:1 or 4, wherein the host cell has been transformed with a polynucleotide encoding said RDH22 homolog.
      • Embodiment 8: Retinol-producing host cell of embodiment 7, wherein the RDH22 homolog is obtained from Yarrowia or Wickerhamomyces.
      • Embodiment 9: Use of the retinol-producing host cell of embodiment 7 or 8 in a process according to embodiment 1, 2, 3, 4, 5, or 6.
      • Embodiment 10: Use of a RDH22 homolog with at least about 30% identity to a polynucleotide according to SEQ ID NO:1 or 4 in a process according to embodiment 1, 2, 3, 4, 5, or 6, wherein the RDH22 homolog is expressed in a suitable retinol-producing host cell.
      • Embodiment 11: Use of RDH22 homolog having activity of YlRDH22 according to SEQ ID NO:1 in a fermentative production of a retinoid-mix with at least about 80% retinyl acetate within said mix using a suitable retinyl acetate producing host cell, wherein the percentage of retinal within said mix and accumulated during said process is reduced to less than 1% based on total retinoids and measured at the end of the production process.
      • Embodiment 12: Use of RDH22 homolog having activity of WaRDH22 according to SEQ ID NO:4 in a fermentative production of a retinoid-mix with at least about 80% retinyl acetate within said mix using a suitable retinyl acetate producing host cell, wherein the percentage of retinal within said mix and accumulated during said process is reduced to about 2% or less based on total retinoids and measured at the end of the production process.
      • Embodiment 13: Process according to embodiment 1, 2, 3, 4, 5, or 6, wherein a host cell according to embodiment 7 or 8 is cultivated in the presence of a lipophilic solvent.
      • Embodiment 14: Process according to embodiment 13, wherein the retinoid mix is accumulated in the lipophilic solvent and optionally extracted from the lipophilic solvent.
  • The following examples are illustrative only and are not intended to limit the scope of the invention in any way. The contents of all references, patent applications, patents, and published patent applications, cited throughout this application are hereby incorporated by reference, particularly WO2019057999, WO2021009194, WO2020141168, WO2019058000, WO2019058001, WO2006102342, WO2008042338, WO2021136689, WO2014096992, WO2016172282.
    • EXAMPLES Example 1: General Methods and Plasmids
  • All basic molecular biology and DNA manipulation procedures described herein are generally performed according to Sambrook et al. (eds.), Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press: New York (1989) or Ausubel et al. (eds). Current Protocols in Molecular Biology. Wiley: New York (1998).
  • Shake plate assay. Typically, 200 μl of 0.075% Yeast extract, 0.25% peptone (0.25X YP) is inoculated with 10 μl of freshly grown Yarrowia and overlaid with 200 μl of Isopar M with 2% oleic acid as a carbon source. Clonal isolates of transformants were grown in 24 well plates (Multitron, 30° C., 800 RPM) in YPD media with Isopar M overlay indicated earlier for 4 days. The overlay fraction was removed from the shake plate wells and analyzed by HPLC on a normal phase column, with a photo-diode array detector.
  • DNA transformation. Strains were transformed by overnight growth on YPD plate media; 50 μl of cells were scraped from a plate and transformed by incubation in 500 μl with 1 μg transforming DNA, typically linear DNA for integrative transformation, 40% PEG 3550MW, 100 mM lithium acetate, 50 mM Dithiothreitol, 5 mM Tris-Cl pH 8.0, 0.5 mM EDTA for 60 minutes at 40° C. and plated directly to selective media or in the case of dominant antibiotic marker selection the cells were out grown on YPD liquid media for 4 hours at 30° C. before plating on the selective media. Nourseothricin (Nat) selection was performed on YPD media containing 100 μg/mL nourseothricin and hygromycin (Hyg) selection was performed on YPD containing 100 μg/mL hygromycin. URA3 marker recycling was performed using 5-fluoroorotic acid (FOA). Episomal hygromycin resistance marker (Hyg) plasmids were cured by passage on non-selective media, with identification of Hyg-sensitive colonies by replica plating colonies from non-selective media to hygromycin containing media (100 μg/mL).
  • DNA molecular biology. Plasmid MB9523 containing expression systems for DrBCO, LmATF-S480Q_G409A_V4071_H69A_1484L, and FfRDH (SEQ ID NO:8) was synthesized at Genscript (Piscataway, NJ, USA). Plasmid MB9523 contains the ‘URA3’ for marker selection in Yarrowia lipolytica transformations. Genes were synthesized with Nhel and Mlul ends in pUC57 vector. Typically, the genes were subcloned to the MB5082 ‘URA3’, MB6157 HygR, and MB8327 NatR vectors for marker selection in Yarrowia lipolytica transformations, as in WO2016172282. For clean gene insertion by random nonhomologous end joining of the gene and marker Hindlll/Xbal (for MB5082-based vectors (all marked with URA3 in Table 1, except MB9523), Pvull (for MB6157—and MB8327-based vectors), or Sfil (for MB9523), respectively purified by gel electrophoresis and Qiagen gel purification column. Clones were verified by sequencing. Typically, genes are synthesized by a synthetic biology at GenScript (Piscataway, NJ). Plasmid MB8388-LIP8 (SEQ ID NO: 9), containing a Cas9, and guide RNA expression systems to target LIP8, was synthesized at Genscript (Piscataway, NJ, USA).
  • Plasmid list. Plasmid, strains, nucleotide and amino acid sequences that were used are listed in Table 1, 2, 6 and the sequence listing. In general, all non-modified sequences referred to herein are the same as the accession sequence in the database for reference strain CLIB122 (Dujon B, et al, Nature. 2004 Jul. 1;430 (6995): 35-44).
  • TABLE 1
    list of plasmids used for construction of the strains for overexpression
    or deletion of the respective genes indicated as “insert”. “LmATF1-mut” refers to
    Lachancea mirantina (LmATF1; SEQ ID NO: 13 in WO2019058001) carrying aa
    substitutions S480Q_G409A_V4071_H69A_1484L. “DrBCO” refers to BCO originated
    from Danio rerio (see SEQ ID NO: 16 in WO2020141168); “FfRDH″ refers to RDH
    originated from Fusarium (see SEQ ID NO: 22 in WO2020141168). For more
    explanation, see text.
    SEQ
    Plasmid Insert Marker ID NO:
    MB7452 Cas9 Nat 7
    MB9523 DrBCO; LmATF1-mut; FfRDH URA3 8
    MB8388-LIP8 Cas9; LIP8 targeting guide RNAs Hyg 9
    MB8203 Homo sapiens RDH12 (HsRDH12) URA3 10
    MB9894 YALIOF29821p (YIRDH22) URA3 11
    MB9997 Wickerhamomyces anomalus RDH22 (WaRDH22) URA3 12
  • TABLE 2
    list of Yarrowia lipolytica strains used. Construction of ML17544 is
    described in Table 2 of WO2020141168. For more details, see text.
    Strain Description
    ML18812 ML17544 transformed with MB9523
    ML18812-lip8 ML18812 with MB7542 and MB8388-LIP8, lip8 deletion
    ML18812-lip8 + MB9894 ML18812-lip8 cured on FOA with MB9894
    ML18812-lip8 + MB8203 + MB9894 ML18812-lip8 + MB8203 cured on FOA, transformed
    with MB9894
  • Normal phase retinol method. A Waters 1525 binary pump attached to a Waters 717 auto sampler were used to inject samples. A Phenomenex Luna 3u Silica (2), 150×4.6 mm with a security silica guard column kit was used to resolve retinoids. The mobile phase consists of either, 1000 mL hexane, 30 mL isopropanol, and 0.1 mL acetic acid for astaxanthin related compounds, or 1000 mL hexane, 60 mL isopropanol, and 0.1 mL acetic acid for zeaxanthin related compounds. The flow rate for each was 0.6 mL per minute. Column temperature was ambient. The injection volume was 20 μL. The detector was a photodiode array detector collecting from 210 to 600 nm. Analytes were detected according to Table 3.
  • TABLE 3
    list of analytes using normal phase retinol method. “RT” means
    retention time. For more details, see text.
    Lambda max
    Intermediates RT [min] [nm]
    11-cis-dihydro-retinol 7.1 293
    11-cis-retinal 4 364
    11-cis-retinol 8.6 318
    13-cis-retinal 4.1 364
    dihydro-retinol 9.2 292
    retinyl-acetate 3.5 326
    retinyl-ester 3 325
    trans-retinal 4.7 376
    trans-retinol 10.5 325
  • Sample preparation. Samples were prepared by various methods depending on the conditions. For whole broth or washed broth samples the broth was placed in a Precellys® tube weighed and mobile phase was added, the samples were processed in a Precellys® homogenizer (Bertin Corp, Rockville, MD, USA) on the highest setting 3X according to the manufactures directions. In the washed broth the samples were spun in a 1.7 ml tube in a microfuge at 10000 rpm for 1 minute, the broth decanted, 1 ml water added mixed pelleted and decanted and brought up to the original volume the mixture was pelleted again and brought up in appropriate amount of mobile phase and processed by Precellys® bead beating. For analysis of mineral oil fraction, the sample was spun at 4000 RPM for 10 minutes and the oil was decanted off the top by positive displacement pipet (Eppendorf, Hauppauge, NY, USA) and diluted into mobile phase mixed by vortexing and measured for retinoid concentration by HPLC analysis.
  • Fermentation conditions. Fermentations were identical to the previously described conditions using Isopar M overlay and stirred tank that was ethanol fed in a bench top reactor with 0.5L to 5L total volume (see WO2016172282). Generally, the same results were observed with a fed batch stirred tank reactor with an increased productivity demonstrating the utility of the system for the production of retinoids.
    • Example 2: Production of Retinoids in Yarrowia lipolytica
  • Strains were grown in the shake plate assay described in Example 1. Addition of plasmids MB9894 (SEQ ID NO:11) or MB9997 (SEQ ID NO:12) to strain ML18812-lip8 improved total retinoid production, when compared to the parental strain which did not contain YlRDH22 or WaRDH22 expression cassettes. Addition of the HsRDH12 expression plasmid MB8203 (SEQ ID NO:10) into strain ML18812-lip8+MB9894 (which results in strain ML18812-lip8+MB8203+MB9894) resulted in a further increased retinoid output. In addition, introduction of MB9894 or MB9997 to ML18812-lip (to make ML18812-lip8+MB9894) reduced the retinal content. The strain ML18812-lip8+MB9894+MB8203 displayed a further reduction of the relative retinal content of the retinoids, and improved the purity of the retinyl acetate, when compared to the parental strain which did not have them. The results are shown in Table 4.
  • TABLE 4
    retinoid production in strain ML18812-lip8 (see Table 2)
    as control compared to said strain carrying plasmids
    as indicated. “% total retinoids” is the
    percentage of retinoids produced compared
    to total retinoids in the control
    without addition of plasmids, wherein
    the total retinoids obtained with the
    control is set to 100%. “% retinal” means
    percentage of retinal in the retinoid mix based
    on total retinoids. For more explanation, see text.
    % total %
    Strain retinoids retinal
    ML18812-lip8 100 8.2
    ML18812-lip8 + MB9894 114.6 0.95
    ML18812-lip8 + MB8203 + MB9894 136 0.6
    ML18812-lip8 + MB9997 110 2.0
    • Example 3: RDH Expression Diminishes Dihydroretinoid Production
  • Strains were fermented as described in Example 1, and broth samples were quantified for dihydroretinoid compounds, the results are shown in Table 5. Addition of either HsRDH12 (MB8203), WaRDH22 (MB9997), or YlRDH22 to ML18812-lip8 reduces both retinal and dihydroretinoid fraction, while adding both HsRDH12 and YlRDH22 (MB9894) reduces it further still. Also, addition of either HsRDH12, WaRDH22, YlRDH22, or both HsRDH12 and YlRDH22 increases retinyl acetate purity.
  • TABLE 5
    retinoid production in strain ML18812-lip8
    (see Table 2) as control compared to said strain carrying
    plasmids as indicated, including the purity with
    regards to retinyl acetate, retinal and
    dihydroretinoids. The percentages given
    are based on total retinoids in the mixture.
    For more explanation, see text.
    % retinyl % dihydro-
    Strain acetate retinoids
    ML18812-lip8 73 10.2
    ML18812-lip8 + MB9894 83 0.3
    ML18812-lip8 + MB8203 + MB9894 92 0.6
    ML18812-lip8 + MB9997 91 1.98
  • TABLE 6
    list of sequences including protein and polynucleotide sequences.
    Nucleotide sequences with a ″*″ refer to codon-optimized sequences. ″aa″ refers
    to amino acids sequence, ″nt″ refers to nucleotide sequence. For more details,
    see text.
    SEQ
    ID
    Name Sequence 5′ to 3′ NO:
    YlRDH22 MTRSKFTEYKQVLRNSCVGKPQYTEKDYPDLDGKNFLVTGATGGVGLEATKLL  1
    (aa) LEKKSHVIMVGRSKTKSQSTLDELOKTYSHGTFDFVEADLSDLTTVERAGEYI
    RSKYTTLDGAILNAGVMAPPYSLTPQGHESQWGINVVAHFLLSKYISPALISA
    AQTAPKDTVRLVWVSSSVVAMSPYEGGIKFDDINHSKVKNPSPWTLYSQSKIG
    DAYLAYLWSKHHPDSGVLSVSLDPGNLASDLSRHTSWLSSIKNYVLYPPKYGA
    YTELSALLNPSVKNNEHLIPWGIEGHLRQDVDDGRRGKDGEELWQGLNKDVEG
    FFKEE
    YlRDH22 ATGACTCGATCCAAGTTCACCGAATACAAACAAGTGCTGCGAAACAGCTGCGT  2
    (nt) GGGCAAGCCCCAATACACCGAGAAGGATTACCCTGATCTCGACGGAAAGAACT
    TTCTTGTCACTGGAGCCACTGGAGGAGTTGGTCTGGAGGCCACCAAGCTGCTG
    CTAGAGAAGAAGAGCCATGTAATCATGGTTGGCCGATCCAAAACAAAGTCTCA
    GTCTACCCTTGACGAGCTCCAGAAGACATACTCCCACGGTACCTTTGATTTCG
    TCGAGGCTGATCTGTCTGACCTGACCACTGTGGAGCGAGCCGGAGAGTACATT
    CGAAGTAAGTACACCACTTTGGACGGTGCCATTCTCAACGCTGGAGTCATGGC
    TCCCCCCTATAGCCTCACTCCCCAGGGCCACGAGTCTCAATGGGGTATCAATG
    TGGTTGCCCATTTTCTGCTGTCCAAGTATATCTCTCCTGCTCTGATCTCGGCC
    GCCCAGACAGCCCCCAAGGACACCGTGCGACTTGTCTGGGTGTCTTCTTCTGT
    CGTTGCCATGAGTCCTTATGAAGGAGGTATCAAGTTTGACGATATCAACCACT
    CTAAGGTCAAGAACCCTTCTCCCTGGACCCTTTACTCCCAGTCCAAGATTGGA
    GATGCTTACCTGGCTTACCTGTGGTCGAAACACCATCCCGATTCTGGCGTCCT
    GTCCGTCTCTCTGGACCCCGGTAACCTGGCCTCTGATCTGTCCAGACATACTT
    CTTGGCTCTCCAGCATTAAGAACTACGTGCTGTATCCTCCCAAGTATGGCGCC
    TACACCGAGCTGTCTGCTCTGCTGAACCCCTCCGTCAAGAATAACGAGCATCT
    CATTCCCTGGGGTATTGAGGGACACCTTAGACAGGATGTCGACGATGGACGAC
    GTGGAAAGGACGGAGAAGAGCTCTGGCAGGGTCTCAACAAGGATGTCGAGGGC
    TTCTTTAAGGAGGAGTAA
    YlRDH22 ATGACCCGATCCAAGTTCACCGAGTACAAGCAGGTTCTCCGAAACTCT  3
    (nt*) TGTGTTGGCAAGCCCCAGTACACTGAGAAGGATTACCCCGATCTCGAC
    GGAAAGAACTTTCTCGTCACTGGTGCTACTGGTGGAGTTGGTCTGGAG
    GCCACTAAGCTGCTCCTTGAGAAGAAGTCTCACGTTATTATGGTTGGC
    CGATCTAAGACTAAGTCTCAGTCTACCCTTGACGAGCTTCAGAAGACT
    TACTCTCACGGTACTTTCGATTTCGTTGAGGCTGATCTCTCTGACCTT
    ACCACTGTTGAGCGAGCCGGAGAGTACATTCGATCTAAGTACACTACC
    CTTGACGGTGCCATTCTTAACGCTGGAGTCATGGCTCCCCCCTACTCT
    CTCACTCCCCAGGGTCACGAGTCTCAGTGGGGTATTAACGTTGTTGCT
    CACTTTCTCCTGTCTAAGTACATCTCTCCTGCTCTGATTTCTGCTGCT
    CAGACTGCCCCCAAGGACACCGTTCGACTTGTTTGGGTGTCTTCTTCT
    GTCGTTGCCATGTCTCCTTACGAGGGAGGTATTAAGTTTGACGATATT
    AACCACTCTAAGGTCAAGAACCCTTCTCCTTGGACCCTTTACTCTCAG
    TCTAAGATTGGTGATGCTTACCTGGCTTACCTTTGGTCTAAGCACCAC
    CCCGATTCTGGAGTCCTTTCTGTCTCTCTGGACCCCGGTAACCTGGCT
    TCTGATCTCTCTCGACACACTTCTTGGCTTTCTTCTATTAAGAACTAC
    GTTCTTTACCCTCCCAAGTACGGTGCCTACACTGAGCTGTCTGCTCTT
    CTCAACCCTTCCGTTAAGAACAACGAGCACCTTATTCCCTGGGGTATT
    GAGGGACACCTTCGACAGGATGTCGACGATGGTCGACGAGGAAAGGAC
    GGAGAGGAGCTCTGGCAGGGTCTTAACAAGGATGTCGAGGGTTTCTTT
    AAGGAGGAGTAA
    WaRDH22 MVNVAYEWLRDCWPPKPEFLPKDYPDLTGKTILITGANTGIGFESAKA  4
    (aa) LLKKNATVVFANRSEEKTKIAIAKIQAELGGDSSQRSIFVKTDLSDLS
    SIKGTAEELKSKGITKLHYTILNAGVMQPPKGSKTKQGFELQIGTNVL
    GHQLLQKFLTPLVLNAVSSDFTPRVVWLASAAHLSSPPNGGIDWDSER
    NADWAGTVSAYGQSKTGNIYQAYIYAQQHKDVISVSAHPGYLASDLTR
    AYKGYLQYLMKLLMSPPVYGSYTELFAALSPKVTLKESGRYIGPWGOF
    RELRDDVQEGLTDGTAQKLWDWAEAEIRPYT
    WaRDH22 ATGGTTAACGTTGCTTATGAATGGCTAAGAGACTGCTGGCCTCCTAAACCTGA  5
    (nt) ATTCTTACCAAAAGATTATCCAGATTTAACAGGAAAGACAATCTTAATCACTG
    GTGCCAATACGGGTATTGGGTTTGAAAGTGCTAAAGCCTTGTTGAAGAAAAAC
    GCAACAGTTGTATTTGCAAATAGAAGCGAAGAGAAGACAAAAATCGCTATTGC
    CAAAATTCAAGCTGAATTAGGTGGTGATTCATCTCAAAGATCAATTTTCGTCA
    AGACTGATTTAAGTGATTTATCATCGATCAAGGGCACAGCGGAAGAATTGAAA
    TCAAAAGGTATCACAAAATTACATTACACCATATTGAATGCTGGTGTTATGCA
    ACCTCCAAAAGGTTCCAAGACAAAACAAGGATTTGAATTACAAATTGGTACAA
    ATGTTTTAGGTCATCAATTATTACAAAAATTTCTAACACCATTAGTTTTGAAT
    GCAGTTTCATCAGATTTCACACCAAGAGTTGTTTGGTTAGCCTCAGCTGCACA
    TCTTTCATCCCCACCAAATGGTGGTATTGATTGGGATTCCTTCCGTAATGCTG
    ATTGGGCTGGAACTGTCAGTGCTTATGGTCAATCCAAGACTGGTAATATTTAT
    CAAGCTTATATCTATGCTCAGCAACATAAAGATGTCATCAGTGTCTCTGCTCA
    TCCAGGGTATTTGGCTTCTGATTTAACAAGAGCTTATAAAGGTTATCTTCAAT
    ATTTGATGAAACTCTTGATGTCTCCTCCAGTTTATGGTTCTTATACTGAATTA
    TTCGCTGCTCTCAGTCCAAAAGTTACATTGAAGGAAAGTGGTAGATATATTGG
    TCCTTGGGGTCAATTCAGAGAATTGAGAGATGATGTTCAAGAAGGCTTAACTG
    ATGGAACTGCTCAAAAATTATGGGATTGGGCTGAAGCTGAAATTAGACCATAC
    ACTTAG
    WaRDH22 ATGGTTAACGTTGCTTACGAGTGGCTTCGAGACTGCTGGCCCCCTAAGCCTGA  6
    (nt*) GTTCCTCCCTAAGGATTACCCTGACCTCACCGGAAAGACCATCCTTATCACTG
    GTGCCAACACTGGTATTGGTTTTGAGTCCGCTAAGGCTCTCCTCAAGAAGAAC
    GCTACCGTTGTTTTTGCCAACCGATCCGAGGAGAAGACTAAGATCGCTATTGC
    CAAGATTCAGGCTGAGCTTGGTGGTGATTCTTCTCAGCGATCTATTTTCGTCA
    AGACTGACCTTTCCGATCTCTCTTCCATTAAGGGCACTGCTGAGGAGCTTAAG
    TCTAAGGGTATCACTAAGCTCCACTACACCATTCTTAACGCTGGTGTTATGCA
    GCCTCCCAAGGGCTCCAAGACTAAGCAGGGTTTTGAGCTTCAGATTGGTACTA
    ACGTTCTTGGCCACCAGCTCCTCCAGAAGTTTCTCACTCCCCTTGTTCTCAAC
    GCTGTTTCTTCTGATTTCACCCCTCGAGTTGTTTGGCTCGCCTCTGCTGCTCA
    CCTTTCTTCCCCTCCCAACGGTGGTATTGATTGGGATTCCTTCCGAAACGCTG
    ATTGGGCTGGTACTGTCTCTGCTTACGGTCAGTCTAAGACTGGTAACATTTAC
    CAGGCTTACATCTACGCTCAGCAGCACAAGGATGTCATCTCTGTCTCTGCTCA
    CCCCGGATACCTTGCTTCTGATCTCACTCGAGCTTACAAGGGATACCTTCAGT
    ACCTTATGAAGCTCCTTATGTCTCCTCCCGTTTACGGTTCTTACACTGAGCTT
    TTCGCTGCTCTTTCTCCCAAGGTTACTCTCAAGGAGTCTGGTCGATACATTGG
    TCCTTGGGGTCAGTTCCGAGAGCTTCGAGATGACGTTCAGGAGGGCCTTACCG
    ATGGTACTGCTCAGAAGCTCTGGGATTGGGCTGAGGCTGAGATTCGACCTTAC
    ACTTAA
    MB7452 CGCGTGGATCGCCGGTGCGTTGACGTTGGTGACCTCCAGCCAGAGGTGCCCGG  7
    CGCCCCGCTCGCCGGCGAACTCCGTCGCGAGCCCCATCAACGCGCGCCCGACC
    CCGTGCCCCCGGTGCTCCGGGGCGACCTCGATGTCCTCGACGGTCAGCCGGCG
    GTTCCACGCCGAGTACGAGATGACCACGAAGCCCGCCAGGTCGCCGTCGTCCC
    CGTACGCGACGAACGTCCGGGAGTCCGGGTCGCCGTCCTCCCCGTCGTCCGAT
    TCGTCGTCCGATTCGTCGTCGGGGAACACCTTGGTCAGGGGCGGGTCCACCGG
    CACCTCCCGCAGGGTGAAGCCGTCCCCGGTGGCGGTGACGCGGAAGACGGTGT
    CGGTGGTGAAGGACCCATCCAGTGCCTCGATGGCCTCGGCGTCCCCCGGGACA
    CTGGTGCGGTACCGGTAAGCCGTGTCGTCAAGAGTGGTCATTTTTGTGTCTAG
    GTGTTTGTGTTTGGACTGCGATCAGTGAAGAAAAGAAGAGGAAAAATTGTGCA
    AGAAATTTTGCTTTCAAGACTTGGCTGATGCAGCAGGGTAACTCTGGGACACA
    GACCTATGTTTGTGGTTAAACTCAATGCACGTGGTACGTGCGTGGAGCGCTTA
    CCCATCCAAGGGTGTGGACATGGAACCGACGGTCCGTGGAGTTGTGTAATGTC
    ATTTTGGCGACTCTTGAAGCAAGGCTATAAAAAAATTGTGTGGCTTGAGTCTT
    ATCGAGCTCGGTCACTACAAGAGTTAATCTTCCTGTCTCAGGCAGACAGGTCA
    GGCAGGGTTACTTTTGGGTGTGCTGTAACTCACTGTATGGCCGTTAGTGCGCA
    TAGACGTTGTACATACTGGACCGAATTGTAGCGTGCTCAATAGGGCCAATAAA
    GCTATTGTAGGGATCCGAATTTTCAGAACCTAATTTATCTGTTACCCGGCCTG
    TGGCTCGCACAGCTTAAAAATGGTCAAACTTTCCCCTTCTTGTCTTTTTTTCC
    TCACATTCATCAGGTTCTTGTCTTGATCTTTCAAGTGAGTATTAATTACCGAC
    CTTGGTTCTTCATTGGGAGAGCATTGGAAGCCGTGGTGCAGCAACCACAAAAC
    GGTTCTTCCCCTTCGATACCTTCTTGCCTGCCTTTCAATACAAGTCGGCTCGA
    TTAGCGGTGGTCGCCCCCGCCAGCGGAGAACATGGAACTAACCCAGAATGAGA
    GCTAAGTGGAGAAAGAAGAGAGTCAGACGACTCAAGCGAAAGCGCCGCAAGGT
    CCGAGCTCGATCCAAATAAGCGGTTTTTAACGGAGATTTAACACTAAATCGAA
    GAACTTTTCCCGTTTCATTTGCGAATGAGCTCGTTAACAAAATCCCCCAGTTT
    TTTTATCCAGCTGTAAGGATTGACATTAGTAATGAATTATTGTTTGGTATATT
    TAAATCTGTAGTTCCTTTCTGTCCGTGTCGGCAACTGTCGTACTCGTGATTTA
    CTTGTATTGACGAATACTTACTGTAGCGCACTCTGCTGCTACTGGTCGTAAGG
    ATGTGCTATTTCGGTGTATGGTGGGTTTTTTGGGGGTCGGAACCGAAGACTGT
    TACACGGGCACGGCTCGTTGTGTACACGCACAGAGCTCTTGCGAGTCATGTTG
    TAGCTAGCTCGTCGTGTTCAGGAACTGTTCGATGGTTCGGAGAGAGTCGCCGC
    CCAGAACATACGCGCACCGATGTCAGCAGACAGCCTTATTACAAGTATATTCA
    AGCAAGTATATCCGTAGGGTGCGGGTGATTTGGATCTAAGGTTCGTACTCAAC
    ACTCACGAGCAGCTTGCCTATGTTACATCCTTTTATCAGACATAACATAATTG
    GAGTTTACTTACACACGGGGTGTACCTGTATGAGCACCACCTACAATTGTAGC
    ACTGGTACTTGTACAAAGAATTTATTCGTACGAATCACAGGGACGGCCGCCCT
    CACCGAACCAGCGAATACCTCAGCGGTCCCCTGCAGTGACTCAACAAAGCGAT
    ATGAACATCTTGCGATGGTATCCTGCTGATAGTTTTTACTGTACAAACACCTG
    TGTAGCTCCTTCTAGCATTTTTAAGTTATTCACACCTCAAGGGGAGGGATAAA
    TTAAATAAATTCCAAAAGCGAAGATCGAGAAACTAAATTAAAATTCCAAAAAC
    GAAGTTGGAACACAACCCCCCGAAAAAAAACAACAAACAAAAAACCCAACAAA
    ATAAACAAAAACAAAATAAATATATAACTACCAGTATCTGACTAAAAGTTCAA
    ATACTCGTACTTACAACAAATAGAAATGAGCCGGCCAAAATTCTGCAGAAAAA
    AATTTCAAACAAGTACTGGTATAATTAAATTAAAAAACACATCAAAGTATCAT
    AACGTTAGTTATTTTATTTTATTTAATAAAAGAAAACAACAAGATGGGCTCAA
    AACTTTCAACTTATACGATACATACCAAATAACAATTTAGTATTTATCTAAGT
    GCTTTTCGTAGATAATGGAATACAAATGGATATCCAGAGTATACACATGGATA
    GTATACACTGACACGACAATTCTGTATCTCTTTATGTTAACTACTGTGAGGCA
    TTAAATAGAGCTTGATATATAAAATGTTACATTTCACAGTCTGAACTTTTGCA
    GATTACCTAATTTGGTAAGATATTAATTATGAACTGAAAGTTGATGGCATCCC
    TAAATTTGATGAAAGATGAAATTGTAAATGAGGTGGTAAAAGAGCTACAGTCG
    TTTTGTTTTGAGATACCATCATCTCTAACGAAATATCTATTAAAAATCTCAGT
    GTGATCATGAGTCATTGCCATCCTGGAAAATGTCATCATGGCTGATATTTCTA
    ACTGTTTACTTGAGATAAATATATATTTACAAGAACTTCCCTTGAAATTAATT
    TAGATATAAAATGTTTGCGGGCAAGTTACTACGAGGAATAAATTATATCTAGA
    GGTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCG
    AGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGG
    ATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGT
    AAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCA
    TCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAA
    GATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACC
    CTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCT
    TTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCA
    AGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCC
    GGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGC
    AGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAG
    AGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGT
    ATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTG
    ATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGC
    AGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACG
    GGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAG
    ATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTA
    AATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTA
    ATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGC
    CTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCC
    CCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCA
    GCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTT
    ATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTT
    CGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTGCAGGCATCGTGGTG
    TCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAG
    GCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTC
    CTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATG
    GCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGT
    GACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGA
    GTTGCTCTTGCCCGGCGTCAACACGGGATAATACCGCGCCACATAGCAGAACT
    TTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGAT
    CTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGAT
    CTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGG
    CAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCAT
    ACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGA
    GCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGC
    ACATTTCCCCGAAAAGTGCCACCTGACGTCTAAGAAACCATTATTATCATGAC
    ATTAACCTATAAAAATAGGCGTATCACGAGGCCCTTTCGTCTGGCCTAGGAAG
    CGACTTCCAATCGCTTTGCATATCCAGTACCACACCCACAGGCGTTTGTGCTA
    CTCTACTGATAGCAATAGATGCGTCATAATTGGTTGGCCCGCTGAGCCTCCAC
    AGGATACTATTGCACATACCCTGGTCATGTGCAGATCAGCTCATTTGTGGAGA
    CTCTGGAGTAACTTAGACGACGCCTGGTTCAATTGCCGCAATGTGCGCCCACG
    CAGATAATGTATTGAGGGGTGGAGCGCCTCTTGGGGACTTGCTGTACTTGTAC
    GGGATATTAAACGCACTCAGCAAGACCATGACGTAAAACACACCTACTGTACG
    ATACGTACTGTAGGTATTGTACTCGTACCCGGTACTACAAATAGTACGATACT
    ATACGGAGTGTATTTGTACCTTGATATACGACTGGCGGAGTGAAGAGAAGGAG
    TTGAACAAGACCAGATGGGGATATCAGCCCCAGTGCTTTGTATTACAAGTACG
    AGTACTTAATAGATACTGTAAGGCTATTGATACGGATGGCAGTAAGTCATTGA
    GTAAGCAATTGTGGCCCAGCATCTCCCCTACGTACTTGTACCATACCCCATGG
    AGACACCAATGGTCTTTCACGCACACTGTCGTGTGCTGTATCGCAGAATCGGG
    TGTCCAACCAAATGCCGTTACCCCCACGTCACAGCCGATAGACAGATACACCA
    TCAATACCAGCAGGTTGTATCATGCGGTTGGCTGAAGGTAAGCTGATTGGTCT
    AAAAACTGTAGCTGTCCTAATTCAACGAGCGCTATTTGGGGCCAACCACCTCG
    GCCAAGCGGCCTTTAATCTGCGTGCCCCAGAGGCGTCTAATGAGGCTCTGGCC
    GCCACTGTAGGAGTGTTTCTCTGTGCGCACACGCAGTTTTGAGTTTGGGCGAC
    TTTCCCTTTTTCCCAATTGCGTACACACACAGCTCCGAGCTAAGCGCTGTCCT
    TGAACCTTCTCCCTCTTTTCCCTCTTTTTCTCTTCCCCTTCCCCTCCTCCACA
    TTAAGGCCAAATCCTGAATTGCACCAACTAGTACAACGACAACAATGGACAAG
    AAGTACTCCATCGGTTTGGACATTGGTACTAACTCTGTCGGCTGGGCCGTCAT
    CACCGACGAGTACAAGGTTCCCTCCAAGAAGTTCAAGGTCCTTGGCAACACCG
    ACCGACACTCTATCAAGAAGAACCTGATCGGTGCTCTGCTGTTCGACTCTGGC
    GAGACTGCCGAGGCCACCCGACTGAAGCGAACCGCTCGACGCCGATACACCCG
    ACGAAAGAACCGAATCTGTTACCTCCAGGAGATCTTCAGCAACGAGATGGCTA
    AGGTCGACGACTCCTTCTTCCACCGACTCGAGGAGTCTTTCCTGGTCGAAGAG
    GATAAGAAGCACGAGCGACACCCCATCTTCGGCAACATTGTTGATGAGGTTGC
    CTACCATGAGAAGTACCCCACCATCTACCACCTCCGAAAGAAGCTCGTCGACT
    CCACTGACAAGGCTGACCTCCGACTCATCTACCTTGCTCTCGCCCACATGATC
    AAGTTCCGAGGTCACTTCCTCATTGAGGGTGATCTCAACCCCGACAACTCCGA
    CGTTGACAAGCTGTTCATCCAGCTCGTCCAGACCTACAACCAGCTCTTTGAGG
    AGAACCCTATCAACGCTTCTGGTGTTGACGCCAAGGCCATTCTCTCCGCCCGA
    CTCTCTAAGTCCCGACGACTCGAGAACCTCATTGCCCAGCTGCCCGGCGAGAA
    GAAGAACGGCCTCTTCGGTAACCTGATTGCTCTCTCTCTTGGTCTGACCCCCA
    ACTTCAAGTCCAACTTTGACCTCGCCGAGGACGCCAAGCTCCAGCTGTCCAAG
    GACACCTACGATGACGATCTGGACAACCTCCTGGCCCAGATCGGTGACCAGTA
    CGCCGATCTCTTCCTTGCCGCCAAGAACCTCTCCGACGCCATCCTGCTCTCCG
    ACATCCTCCGAGTCAACACCGAGATTACCAAGGCTCCTCTGTCTGCCTCTATG
    ATCAAGCGATACGACGAGCACCACCAGGATCTCACTCTTCTCAAGGCTCTCGT
    CCGACAGCAGCTCCCCGAGAAGTACAAGGAGATTTTCTTTGACCAGTCCAAGA
    ACGGTTACGCTGGCTACATTGACGGTGGTGCTTCCCAGGAAGAGTTTTACAAG
    TTCATCAAGCCTATTCTGGAGAAGATGGACGGTACCGAGGAGCTGCTCGTCAA
    GCTCAACCGAGAGGACCTCCTTCGAAAGCAGCGAACCTTCGATAACGGCTCCA
    TCCCCCACCAGATCCACCTGGGTGAGCTCCACGCCATTCTCCGAAGACAAGAG
    GACTTCTACCCCTTCCTAAAGGATAACCGAGAGAAGATCGAGAAGATTCTCAC
    CTTCCGAATCCCCTACTACGTCGGTCCCCTCGCTCGAGGTAACTCCCGATTTG
    CTTGGATGACCCGAAAGTCCGAGGAGACTATCACCCCCTGGAACTTTGAAGAG
    GTAGTCGACAAGGGTGCCTCCGCCCAGTCTTTCATTGAGCGGATGACCAACTT
    CGATAAGAACCTCCCCAACGAGAAGGTCCTTCCCAAGCACTCTCTCCTCTACG
    AGTACTTCACCGTCTACAACGAGCTGACCAAGGTCAAGTACGTTACCGAGGGC
    ATGCGAAAGCCCGCTTTCCTCTCTGGTGAGCAGAAGAAGGCCATTGTCGACCT
    CCTGTTCAAGACTAACCGAAAAGTCACCGTCAAGCAGCTCAAGGAAGACTACT
    TCAAGAAGATTGAGTGCTTCGACTCCGTCGAGATTTCCGGTGTCGAGGACCGA
    TTCAACGCCTCCCTCGGCACCTACCACGATCTTCTGAAGATCATCAAGGACAA
    GGACTTTCTTGATAACGAGGAGAACGAGGACATTCTCGAGGACATCGTCCTCA
    CCCTCACCCTTTTCGAGGATCGAGAGATGATCGAGGAGCGACTCAAGACCTAC
    GCCCATCTCTTCGACGACAAGGTCATGAAGCAACTCAAGCGACGACGATACAC
    TGGCTGGGGCCGACTTTCCCGAAAGCTCATCAACGGCATCCGAGACAAGCAGT
    CTGGCAAGACCATCCTGGACTTCCTGAAGTCCGACGGTTTCGCCAACCGAAAC
    TTCATGCAGCTCATCCACGACGACTCTCTTACCTTCAAAGAGGATATCCAGAA
    GGCCCAGGTTTCTGGCCAGGGCGACTCCCTCCACGAGCACATTGCCAACCTCG
    CCGGATCCCCCGCCATCAAAAAGGGTATCCTCCAGACCGTCAAGGTTGTCGAC
    GAACTCGTGAAGGTCATGGGCCGACACAAGCCCGAGAACATCGTTATCGAGAT
    GGCCCGAGAGAACCAGACCACCCAGAAGGGTCAGAAGAACTCCCGAGAGCGAA
    TGAAGCGAATCGAAGAGGGTATCAAGGAGCTCGGTTCCCAGATTCTCAAGGAG
    CACCCCGTCGAGAACACCCAGCTCCAGAACGAGAAACTCTACCTGTACTACCT
    CCAGAATGGCCGAGACATGTACGTTGACCAGGAGCTCGACATCAACCGACTCT
    CCGACTACGACGTCGACCACATTGTTCCTCAGTCCTTCCTCAAGGACGACTCC
    ATCGACAACAAGGTTCTGACCCGATCTGACAAGAACCGAGGTAAGTCCGACAA
    CGTTCCCTCCGAAGAGGTCGTTAAGAAGATGAAGAACTACTGGCGACAGCTTC
    TCAACGCCAAACTGATCACCCAGCGAAAGTTTGACAACCTCACCAAGGCCGAG
    CGAGGTGGTCTGTCCGAGCTGGACAAGGCCGGCTTCATTAAGCGACAGCTGGT
    CGAGACTCGACAGATCACCAAGCACGTCGCCCAGATCCTCGACTCCCGAATGA
    ACACCAAGTACGACGAGAACGACAAGCTCATCCGGGAGGTCAAGGTCATCACC
    CTGAAGTCTAAGCTTGTCTCCGACTTCCGAAAGGACTTCCAGTTCTACAAGGT
    CCGAGAGATCAACAACTACCACCACGCCCACGACGCCTACCTCAACGCCGTTG
    TTGGTACCGCCCTCATCAAGAAGTATCCCAAGCTCGAGTCCGAGTTCGTTTAC
    GGCGACTACAAGGTTTACGATGTCCGAAAGATGATTGCCAAGTCCGAGCAGGA
    GATCGGTAAGGCCACCGCCAAGTACTTTTTCTACTCCAACATCATGAATTTCT
    TCAAGACCGAGATCACTCTCGCCAACGGTGAGATTCGAAAGCGACCCCTGATT
    GAGACTAATGGTGAGACTGGTGAGATCGTCTGGGATAAGGGCCGAGACTTCGC
    CACCGTCCGAAAGGTCCTGTCCATGCCCCAGGTCAACATTGTCAAGAAGACCG
    AGGTCCAGACCGGTGGCTTCTCCAAGGAGTCCATTCTCCCCAAGCGAAACTCC
    GACAAACTCATCGCCCGTAAGAAGGACTGGGATCCGAAGAAGTACGGTGGTTT
    CGATTCTCCCACCGTTGCCTACTCCGTCCTCGTTGTTGCTAAAGTCGAGAAGG
    GTAAGTCTAAGAAACTCAAGTCCGTGAAGGAGCTACTCGGTATCACCATCATG
    GAGCGATCTTCTTTTGAGAAGAACCCCATTGACTTCCTCGAGGCCAAGGGTTA
    CAAAGAGGTCAAGAAGGACCTGATTATCAAGCTGCCCAAGTACTCCCTCTTTG
    AGCTCGAGAACGGCCGAAAGCGAATGCTGGCTTCCGCTGGTGAGCTGCAGAAG
    GGCAACGAGCTCGCTCTGCCCTCCAAGTACGTCAACTTCCTCTACCTGGCCTC
    CCACTACGAGAAGCTCAAGGGCTCCCCCGAGGACAACGAGCAGAAGCAGCTGT
    TCGTTGAGCAGCACAAGCACTACCTCGACGAGATCATCGAGCAGATCTCCGAG
    TTCTCCAAGCGAGTCATCCTCGCTGACGCCAACCTTGATAAGGTTCTCTCTGC
    TTACAACAAGCACCGGGACAAGCCCATCCGAGAGCAGGCCGAGAATATCATCC
    ACCTCTTCACTCTCACCAACCTCGGCGCTCCTGCTGCCTTCAAGTACTTCGAC
    ACCACCATTGACCGAAAGAGGTACACCTCCACCAAGGAAGTCCTCGACGCCAC
    CCTGATCCACCAGTCCATCACCGGCCTCTACGAAACCCGAATCGACCTCTCCC
    AGCTCGGCGGTGACTCTCGAGCCGACCCCAAGAAGAAGCGAAAAGTCTAAATA
    TCCGAAGATCAAGAGCGAAGCAAGTTGTAAGTCCAGGACATGTTTCCCGCCCA
    CGCGAGTGATTTATAACACCTCTCTTTTTTGACACCCGCTCGCCTTGAAATTC
    ATGTCACATAAATTATAGTCAACGACGTTTGAATAACTTGTCTTGTAGTTCGA
    TGATGATCATATGATTACATTAATAGTAATTACTGTATTTGATATATATACTA
    ATTACAATAGTACATATTAGAACATACAATAGTTAGTGCCGTGAAGTGGCTTA
    AAATACCGCGAGTCGATTACGTAATATTATTACCTCTTGCCCATCGAACGTAC
    AAGTACTCCTCTGTTCTCTCCTTCCTTTGCTTTGTGCACGAAGAACTGCGGTC
    AGGTGACACAACTTTTTCCATCTCAGGGTGTGTCGCGTGTGCTTCATCCAAAC
    TTTAGTTGGGGTTCGGGTTCGCGCGAGATGATCACGTGCCCTGATTTGGTGTC
    GTCCCCCGTCGCGCTGCGCACGTGATTTATTTATTTCCGGTGGCTGCTGTCTA
    CGCGGGGCCTTCTCTGCCCTTCTGTTTCAACCTTCGGGCGGTTCTCGTAACCA
    GCAGTAGCAATCCATTTCGAAACTCAAAGAGCTAAAAACGTTAAACCTCAGCA
    GTCGCTCGACGAATGGGCTGCGGTTGGGAAGCCCACGAGGCCTATAGCCAGAG
    CCTCGAGTTGACAGGAGCCCAGACGCCTTTTCCAACGGCAACTTTTATATAAA
    ATGGCAATGTATTCATGCAATTGCGGCCGTGTCAGGTTGGAGACACTGGACCA
    CACTCTCCATTGCTTCCTGAGGAGATGGATCATTGCTAGTGCATCTACGCGCA
    GCAATCCCGCAAGCTCGACAACCGTAGATGGGCTTTGGTGGGCCAATCAATTA
    CGCAACCCGCACGTTAAATTGTATGAGGAAGGAAGGCCACGGTACAAAGTGGG
    TGGTCTTCACCCAGTGGTTGTTGGTGGCGTCATGCAGACCATGGCCGCCAGTG
    TGCTGGAATTGAATATTTACCGTTCGTATAATGTATGCTATACGAAGTTATAC
    CGGTCTCGTAGTGTTCACGTTCAGTTCACGGTGAGCTTAAAACTATCTTCAAG
    AAGAGATTTGAGACCTGATTTATACTTGCAGCAATGTTTACTTCTTATCGCGA
    TACACGAATGTGATACGGATCAAAGTAAGCAGGACTACGATAAGATAACGAAT
    GCGGTGCAGTCCATGTCGATTAGGTATAGATACATTTATTTTGTGTTATGTTA
    CATTTTGGGGGGATACTGTCCTACTTGTAGTACCTACTTGTAGTGGCGCGTTA
    GGGGCAGGGCATGCTCATGTAGAGCGCCTGCCGCTCGCCGTCCGAGGCGGTGC
    CGTCGTACAGGGCGGTGTCCAGGCCGCAGAGGGTGAACCCCATCCGCCGGTA
    MB9523 GCGGAGCCTAGGCCGGCCAGCGTGGCGCGTGGCCATATTGGCCAGCAGCTTAC  8
    TACAGCTCAGCCAAAGTGGATCATGATGGACGGAAATATCGGACTGGAGGCCA
    AAAAGGAGGTGCTCAAATACGCACGGGACAAGTCTGCACAGGTGGCATTCGAA
    CCCACGTCTGTCCCCAAAGCCGCTGCTCTTTCCGAGCTCAATCTGCCCGTTTA
    CCCCAACAACTCCATTGCCTTAGCCACCCCCAACACTGCAGAGCTCGAGGCCA
    TGTTCGAGGCATTCCACGAGAAGGGCCGATTTGACGTTGACGACTGGTTTCCA
    GTCATAGACGGTCTTGCTCTCGGAGCTGACTTTAGAAACGGAGCCACCATGTT
    GTCGCACCAACATCGTGGACTCAAAGCTATTCTCGAACAAGGAACTCTGGCCC
    AGGCAATCCACATGCTACCATACATCCCAACTTTGATCATCAAGGGCGGAGCC
    AACGGCGTCGTCGTCTTCCAACTCATTGATGATATCGAGTCTGCCATTCATTC
    ACAGCGTTCTGCTTCCAATAAAACGCCTGGCCTGTTCCAGAAGGGCAACGCTG
    CGAGTGGAAACACCAAGGTCGGCGTCTACATGCAGTACTTTGAGCCCGAAGAA
    GTGGGCAGTCAGTCGATTGTAAGCGTGACTGGAGCTGGAGACACTCTGTTTGG
    AACCCTGGCCATGGAGATTGTCAAGGACGAGTCCTGGTTGAACGATATGGGAG
    ACAAGAAGAGTGCAGTTGTTTCTCGAGCCATGAACAATGCTGTGAAGACTATT
    CAGAGTAAGGACGCTGTCTGCAAGAGCATCCTTTAAGTGATTTGCCATGCTTT
    CTCTTCTTCCACGATGTAAATACTTATTTTACACACTACTGTGCAGTAGCAAA
    TACAGAACAAGAGTTGTCGCCTATTGACAGTACAGTACGAGTAGTGTATGTAC
    AGTAGTTATACAATATCTATGTGAAATTCGTCGGCAGCTTTCGGCTGATGAAC
    TACGAGTTCTTCGTAATAAATCATCAACGTAATAAGCTTGGTACCAGAGACGG
    GTTGGCGGCGTATTTGTGTCCCAAAAAACAGCCCCAATTGCCCCAATTGACCC
    CAAATTGACCCAGTAGCGGGCCCAACCCCGGCGAGAGCCCCCTTCACCCCACA
    TATCAAACCTCCCCCGGTTCCCACACTTGCCGTTAAGGGCGTAGGGTACTGCA
    GTCTGGAATCTACGCTTGTTCAGACTTTGTACTAGTTTCTTTGTCTGGCCATC
    CGGGTAACCCATGCCGGACGCAAAATAGACTACTGAAAATTTTTTTGCTTTGT
    GGTTGGGACTTTAGCCAAGGGTATAAAAGACCACCGTCCCCGAATTACCTTTC
    CTCTTCTTTTCTCTCTCTCCTTGTCAACTCACACCCGAAATCGTTAAGCATTT
    CCTTCTGAGTATAAGAATCATTCGCTAGCCACAAAAATGGGTGATCTCGATGC
    CCGAGGAACCTCTGCTCACCCCGAGCTCTCTGAGCGACCTTCTATTATGCCTT
    CTATGTCTGATATTCAGGACCCTTCTGGTGATGACAAGGCTACTCCCCGAGGA
    TCTGCTGCTGGTCTGCCCCAGCTTGAGCTTGCTGGACACGCCCGACGACTTGG
    CCACCTTGAGAACTTCTTTGCTGTCCAGGCTCGACAGCAGATTTACTCTTCTT
    TTGCTGTTTTTTGTGAGTTTGACACTGCTTGTTCTCTCGCTCAGCTTGCTTCT
    GCTGTGCGAAACGTTTGTCTTTCTAACCCCCTTCTCCTTCACACTGTTGAGCC
    TAAGCACCCTGACATCGCTGGATTCTACCACTCTGACGAGTACCTTTCCCGAC
    CTTGGCCTCAGCACGATTACATGCGAGTTCTTCGAGAGGTTCACGTCGCTGAC
    GTTGTTATGAACGGACAGAAGGAGCACGCTCACGTTGTTCGAGATGCTGTTGA
    CGTTTTTCAGGCTCACGGAAACCAGGTTACTTCTGAGCTCCTTGAGCTTATGA
    CTCAGATTGAGATTCCTCACGCTTCTCAGACTCGACCCTCTTGGCGACTTCTC
    TGTTTTCCCCACGGAGAGGCTAACCGATGGCGAACCTTTGCTTTTGTTTCTAA
    CCACTGTTCTTCTGATGGTCTTTCTGGTCTTAACTTCTTTCGAGATCTCCAGA
    AGGAGCTTGCTCACGGCCCCACCTCTGGTGCTCCTGGTGCCCCCGGAGCTTCC
    GGAGTTATTTTCGATTACGCTCAGGACGCTGCTACCCTGCCCAAGCTGCCCCC
    TCCCATTGATCAGAAGCTCGATTACCGACCTTCTAAGAAGGCTCTTCTCGGCC
    TTCTCGCTGGCAAGTTCGTTCGAGAGAAGCTCGGTTACGTTTCTGCTGCTCCT
    CCCACTACCCCTACCTCTGACCTTGCTCACCCTGAGGGTCACCAGTACTACTG
    TTACCTTGTTAACGTTCCCACTTCTTCTGTTGCCCACATTAAGACTCAGGTGC
    GAGAGAACGTTCCTCACAAGTGTACTCTCACTCCCTTTCTCCAGGCTTGTTGG
    CTTGTTTCTCTGTTCAAGTACGGTCGAGTTTTTTCTGGTTCTTGGCTTGAGCG
    ATACACCGATGTTCTTGTTGCTATGAACACTCGACAGCTTCTCCCCGAGGACC
    TTGAGCTTCAGCGACAGTACCGATACGGTTCTAACATTGGAGCTGTTCGATAC
    AACTACCCTATTGCTCCCCTTGACGTTCGAGATAACGATCAGAAGTTCTGGTC
    CCTTGTTGAGTCTTACCGACTTGCCCTTTCTGATGCCCGAGATAAGAACGATT
    ACCTTTACGCTCTTGGTGCTCTTATGCTCCCTGAGATTTACGAGAAGAAGAAC
    GTTGATGCTGTTGTTAACGATACCATTCTTAACCAGCGACGACAGGGAACCCT
    TCTTTCTAACGTTGGTTACGTTCGAGATGAGCAGCCCACTGCTTTTGCTATTA
    AGAACCACGTTTTTTCTCAGGGAGTTGGAGCTAACCGAAACGCTTTTGTTCTT
    AACATTTGTGCTACCGATCAGGGTGGTCTTAACATCGCTATTTCTATTGCTAA
    GGGAACCCTTGCTTCTCGACAGGAGGGACAGGAGCTTTGTGATATTTTTAAGT
    CTACTCTCCTTCGATTTTAAACGCGTCTATCCGAAGATCAAGAGCGAAGCAAG
    TTGTAAGTCCAGGACATGTTTCCCGCCCACGCGAGTGATTTATAACACCTCTC
    TTTTTTGACACCCGCTCGCCTTGAAATTCATGTCACATAAATTATAGTCAACG
    ACGTTTGAATAACTTGTCTTGTAGTTCGATGATGATCATATGATTACATTAAT
    AGTAATTACTGTATCTGTACCTGCTGTGGACCACGCACGGCGGAACGTACCGT
    ACAAATATTTTCTTGCTCACATGACTCTCTCTCGGCCGCGCACGCCGGTGGCA
    AATTGCTCTTGCATTGGCTCTGTCTCTAGACGTCCAAACCGTCCAAAGTGGCA
    GGGTGACGTGATGCGACGCACGAAGGAGATGGCCCGGTGGCGAGGAACCGGAC
    ACGGCGAGCCGGCGGGAAAAAAGGCGGAAAACGAAAAGCGAAGGGCACAATCT
    GACGGTGCGGCTGCCACCAACCCAAGGAGGCTATTTTGGGTCGCTTTCCATTT
    CACATTCGCCCTCAATGGCCACTTTGCGGTGGTGAACATGGTTTCTGAAACAA
    CCCCCCAGAATTAGAGTATATTGATGTGTTTAAGATTGGGTTGCTATTTGGCC
    ATTGTGGGGGAGGGTAGCGACGTGGAGGACATTCCAGGGCGAATTGAGCCTAG
    AAAGTGGTACCATTCCAACCGTCTCAGTCGTCCGAATTGATCGCTATAACTAT
    CACCTCTCTCACATGTCTACTTCCCCAACCAACATCCCCAACCTCCCCCACAC
    TAAAGTTCACGCCAATAATGTAGGCACTCTTTCTGGGTGTGGGACAGCAGAGC
    AATACGGAGGGGAGATTACACAACGAGCCACAATTGGGGAGATGGTAGCCATC
    TCACTCGACCCGTCGACTTTTGGCAACGCTCAATTACCCACCAAATTTGGGCT
    GGAGTTGAGGGGACCGTGTTCCAGCGCTGTAGGACCAGCAACACACACGGTAT
    CAACAGCAACCAACGCCCCCGCTAATGCACCCAGTACTGCGCAGGTGTGGGCC
    AGGTGCGTTCCAGATGCGAGTTGGCGAACCCTAAGCCGACAGTGTACTTTTTG
    GGACGGGCAGTAGCAATCGTGGGCGTAGACCCCGGTGTATATAAAGGGGTGGA
    GAGGACGGATTATTAGCACCAACACACACACTTATACTACATGCTAGCCACAA
    AAATGCTCTCTTTCTTCTGGCGAAACGGTATCGAGACTCCCGAGCCCCTCAAG
    GCTGACGTTTCCGGCTCTATCCCTCCCTGGCTTCAGGGAACCCTTCTCCGAAA
    CGGTCCTGGTCTGTTCTCCGTTGGCAACACTTCCTACAAGCACTGGTTCGATG
    GTATGGCTCTCATTCACTCCTTCACCTTTAAGGATGGTGAGGTTTTTTACCGA
    TCTAAGTACCTGAAGTCTGAGACTTACAAGAAGAACATCGCTGCCGACCGAAT
    CGTTGTGTCTGAGTTCGGAACCATGGTGTACCCCGATCCCTGCAAGAACATTT
    TCTCCCGAGCCTTCTCTTACATGATGAACGCCATTCCTGACTTTACCGATAAC
    AACCTCATTAACATCATTAAGTACGGTGAGGATTACTACGCCTCCTCTGAGGT
    CAACTACATCAACCAGATTGACCCCCTGACCCTTGAGACTCTCGGACGAACTA
    ACTACCGAAACCACATTGCCATCAACCTTGCCACTGCTCACCCTCACTACGAC
    GAGGAGGGTAACACTTACAACATGGGCACTGCTATTATGAACCTCGGTCGACC
    CAAGTACGTGATTTTCAAGGTGCCCGCCAACACCTCTGATAAGGAGAACAAGA
    AGCCTGCCCTCTCTGAGGTGGAGCAGGTTTGCTCCATTCCCATCCGACCCTCC
    CTTTACCCTTCTTACTTCCACTCTTTTGGCATGACTGAGAACTACATCATCTT
    CGTTGAGCAGGCCTTCAAGCTGGACATCGTCAAGCTGGCTACTGCTTACTTCC
    GAGATATTAACTGGGGATCTTGCCTTAAGTTCGACCAGGATGACATTAACGTG
    TTCCACCTGGTCAACAAGAAGACTGGTAAGGCTGTGTCCGTGAAGTACTACAC
    TGACCCCTTTGTTACCTTCCACCACATCAACGCTTACGAGGACGATGGCCACG
    TCGTCTTCGATCTCATTACTTACAAGGACTCTAAGCTGTACGATATGTTCTAC
    ATTCAGAACATGAAGCAGGACGTCAAGCGATTTATTGAGACTAACAAGGACTT
    CGCTCAGCCCGTGTGCCAGCGATTTGTCCTTCCCGTCAACGTTGATAAGGAGA
    CTCCTCAGGACATCAACCTTGTCAAGCTGCAGGACACCACTGCCACTGCTGTC
    CTGAAGGAGGACGGCTCTGTCTACTGCACCCCTGACATCATTTTTAAGGGTCT
    TGAGCTCCCTGCTATCAACTACAAGTTTAACTCTAAGAAGAACCGATACTTCT
    ACGGCACCCGAGTGGAGTGGTCCCCTTACCCTAACAAGGTCGCTAAGGTGGAC
    GTTGTTACTCGAACCCACAAGATTTGGACTGAGGAGGAGTGTTACCCTTCTGA
    GCCTGTCTTTATTGCCTCCCCTGACGCCGTTGATGAGGATGACGGTGTGATTC
    TTTCTTCTGTGGTTTCTTTCAACCCCCAGCGACCCCCTTTCCTGGTTGTCCTC
    GATGCTAAGTCCTTCAAGGAGATTGCTCGAGCTACCATCGATGCCTCTATTCA
    CATGGACCTTCACGGCCTTTTCATCCACGACAAGTCTACCTAAGTTTTTTGAT
    CAATGATCCAATGGCTTTCACATACCCCCCCACGCCTATAATTAAAACACAGA
    GAAATATAATCTAACTTAATAAATATTACGGAGAATCTTTCGAGTGTTCAGCA
    GAAATATAGCCATTGTAACAAAAGCCGGCTATCGACCGCTTTATCGAAGAATA
    TTTCCCGCCCCCCAGTGGCCAAACGATATCGGTCAGAAGGGGCAGCTCTAAAC
    GAAGAACTGCGGTCAGGTGACACAACTTTTTCCATCTCAGGGTGTGTCGCGTG
    TGCTTCATCCAAACTTTAGTTGGGGTTCGGGTTCGCGCGAGATGATCACGTGC
    CCTGATTTGGTGTCGTCCCCCGTCGCGCTGCGCACGTGATTTATTTATTTCCG
    GTGGCTGCTGTCTACGCGGGGCCTTCTCTGCCCTTCTGTTTCAACCTTCGGGC
    GGTTCTCGTAACCAGCAGTAGCAATCCATTTCGAAACTCAAAGAGCTAAAAAC
    GTTAAACCTCAGCAGTCGCTCGACGAATGGGCTGCGGTTGGGAAGCCCACGAG
    GCCTATAGCCAGAGCCTCGAGTTGACAGGAGCCCAGACGCCTTTTCCAACGGC
    AACTTTTATATAAAATGGCAATGTATTCATGCAATTGCGGCCGTGTCAGGTTG
    GAGACACTGGACCACACTCTCCATTGCTTCCTGAGGAGATGGATCATTGCTAG
    TGCATCTACGCGCAGCAATCCCGCAAGCTCGACAACCGTAGATGGGCTTTGGT
    GGGCCAATCAATTACGCAACCCGCACGTTAAATTGTATGAGGAAGGAAGGCCA
    CGGTACAAAGTGGGTGGTCTTCACCCAGTGGTTGTTGGTGGCGTCATGCAGAC
    CATGCATTGGGGATAGCACAGGGTTGGGGTGTCTTGTGGACTCAATGGGTGAA
    AGGAGATGGAAAAGGGCGGTGAAAAGTGGTAGAATCGAAATCCCTGACGTCAA
    TTTATAAAGTAAAATGCGTTTCTGCCATTTTGCTCCCCTCCTTCTTTCGCAAT
    CGCCTCCCCAAAAGTTGTCGTGGCAGTACACATGCTTGCATACAATGAAGCTA
    ATCCGGCTTGCTCAGTAGTTGCTATATCCAGGCATGGTGTGAAACCCCTCAAA
    GTATATATAGGAGCGGTGAGCCCCAGTCTGGGGTCTTTTCTCTCCATCTCAAA
    ACTACTTTCTCACATGCTAGCCACAAAAATGACCACTAAGTACACTTCCGTTC
    ACGAGTCTCCCAACGGCCCTGGTGACGCTCGACCCACCGCTTCCCAGATTATC
    GACGATTACAACCTTGAGGGAGAGCTTTCTGGCAAGACTGTTCTCGTCACCGG
    CTGTTCCTCTGGTATTGGTGTTGAGACTGCCCGAGCTATTTACCGAACTGGTG
    CCACCCTTTACCTCACTGCCCGAGATGTCGATAAGGCCAAGACCGTTCTTCCC
    GACCTTGTTGACACTTCCCGAGTCCACTTTCTCCACCTTGACCTTAACTCTCT
    GGAGTCTGTTCGAGGTTTTGCTGAGAACTTCAAGTCTAAGTCCACTCAGCTTC
    ACATTCTCATCGAGAACGCTGGCGTGATGGCCTGTCCCGAGGGCCGAACCGTC
    GATGGTTTTGAGACTCAGTTTGGTATCAACCACCTTGCTCACTTTCTCCTCTT
    TTACCTCCTCAAGGATACCCTTCTCAACTCTTCTACCCCCGCTTTCAACTCCC
    GAGTTGTCATCCTCTCTTCTTGTGCTCACCAGGCTGGTTCCGTTCACCTTAAC
    AACCTGTCTCTTGAGGGTGGATACGAGCCTTGGAAGTCTTACGGCCAGTCCAA
    GACTGCCAACCTTTGGACTGCCCGAGAGATCGAGAAGCGATTTGGTGCTTCCG
    GTATCCACTCTTGGGCTGTTCACCCCGGTTCCATCGCTACTGAGCTTCAGCGA
    CACGTTTCCGACGAGCTTAAGCAGAAGTGGGCTGACGATAAGGAGGGTGCCAA
    GCTGTGGAAGTCCACCGAGCAGGGTGCCGCCACCACTGTCCTTGCTGCTGTTT
    CCCCTGAGCTTGAGGGTAAGGGCGGTCTTTACCTTGAGGATACCCAGGTTGCC
    AAGCCCCCTGCCCGAGGAATGTTTGGTGTTGCTGACTGGGCTTACGATGAGGA
    TGGCCCCTCTAAGCTCTGGGCCAAGTCTCTTGAGCTCCTTAAGCTCCAGTAAA
    GGTTAGACTATGGATATGTAATTTAACTGTGTATATAGAGAGCGTGCAAGTAT
    GGAGCGCTTGTTCAGCTTGTATGATGGTCAGACGACCTGTCTGATCGAGTATG
    TATGATACTGCACAACCTGTGTATCCGCATGATCTGTCCAATGGGGCATGTTG
    TTGTGTTTCTCGATACGGAGATGCTGGGTACAAGTAGCTAATACGATTGAACT
    ACTTATACTTATATGAGGCTTGAAGAAAGCTGACTTGTGTATGACTTATTCTC
    AACTACATCCCCAGTCACAATACCACCACTGCACTACCACTACACACTAGTGG
    TGTGTTCTGTGGAGCATTCTCACTTTTGGTAAACGACATTGCTTCAAGTGCAG
    CGGAATCAAAAAGTATAAAGTGGGCAGCGAGTATACCTGTACAGACTGTAGGC
    GATAACTCAATCCAATTACCCCCCACAACATGACTGGCCAAACTGATCTCAAG
    ACTTTATTGAAATCAGCAACACCGATTCTCAATGAAGGCACATACTTCTTCTG
    CAACATTCACTTGACGCCTAAAGTTGGTGAGAAATGGACCGACAAGACATATT
    CTGCTATCCACGGACTGTTGCCTGTGTCGGTGGCTACAATACGTGAGTCAGAA
    GGGCTGACGGTGGTGGTTCCCAAGGAAAAGGTCGACGAGTATCTGTCTGACTC
    GTCATTGCCGCCTTTGGAGTACGACTCCAACTATGAGTGTGCTTGGATCACTT
    TGACGATACATTCTTCGTTGGAGGCTGTGGGTCTGACAGCTGCGTTTTCGGCG
    CGGTTGGCCGACAACAATATCAGCTGCAACGTCATTGCTGGCTTTCATCATGA
    TCACATTTTTGTCGGCAAAGGCGACGCCCAGAGAGCCATTGACGTTCTTTCTA
    ATTTGGACCGATAGCCGTATAGTCCAGTCTATCTATAAGTTCAACTAACTCGT
    AACTATTACCATAACATATACTTCACTGCCCCAGATAAGGTTCCGATAAAAAG
    TTCTGCAGACTAAATTTATTTCAGTCTCCTCTTCACCACCAAAATGCCCTCCT
    ACGAAGCTCGAGCTAACGTCCACAAGTCCGCCTTTGCCGCTCGAGTGCTCAAG
    CTCGTGGCAGCCAAGAAAACCAACCTGTGTGCTTCTCTGGATGTTACCACCAC
    CAAGGAGCTCATTGAGCTTGCCGATAAGGTCGGACCTTATGTGTGCATGATCA
    AGACCCATATCGACATCATTGACGACTTCACCTACGCCGGCACTGTGCTCCCC
    CTCAAGGAACTTGCTCTTAAGCACGGTTTCTTCCTGTTCGAGGACAGAAAGTT
    CGCAGATATTGGCAACACTGTCAAGCACCAGTACAAGAACGGTGTCTACCGAA
    TCGCCGAGTGGTCCGATATCACCAACGCCCACGGTGTACCCGGAACCGGAATC
    ATTGCTGGCCTGCGAGCTGGTGCCGAGGAAACTGTCTCTGAACAGAAGAAGGA
    GGACGTCTCTGACTACGAGAACTCCCAGTACAAGGAGTTCCTGGTCCCCTCTC
    CCAACGAGAAGCTGGCCAGAGGTCTGCTCATGCTGGCCGAGCTGTCTTGCAAG
    GGCTCTCTGGCCACTGGCGAGTACTCCAAGCAGACCATTGAGCTTGCCCGATC
    CGACCCCGAGTTTGTGGTTGGCTTCATTGCCCAGAACCGACCTAAGGGCGACT
    CTGAGGACTGGCTTATTCTGACCCCCGGGGTGGGTCTTGACGACAAGGGAGAC
    GCTCTCGGACAGCAGTACCGAACTGTTGAGGATGTCATGTCTACCGGAACGGA
    TATCATAATTGTCGGCCGAGGTCTGTACGGCCAGAACCGAGATCCTATTGAGG
    AGGCCAAGCGATACCAGAAGGCTGGCTGGGAGGCTTACCAGAAGATTAACTGT
    TAGAGGTTAGACTATGGATATGTAATTTAACTGTGTATATAGAGAGCGTGCAA
    GTATGGAGCGCTTGTTCAGCTTGTATGATGGTCAGACGACCTGTCTGATCGAG
    TATGTATGATACTGCACAACCTGTGTATCCGCATGATCTGTCCAATGGGGCAT
    GTTGTTGTGTTTCTCGATACGGAGATGCTGGGTACAAGTAGCTAATACGATTG
    AACTACTTATACTTATATGAGGCTTGAAGAAAGCTGACTTGTGTATGACTTAT
    TCTCAACTACATCCCCAGTCACAATACCACCACTGCACTACCACTACACCTCG
    AGCATGCACACTATTATCACATTACTACATCCAAACCCCTACAGGGGGAGGAG
    CTCTCCAATCAAATATGTACATTAACTATCTCTCGTAAATCATTGTTATAAGA
    CCGTCCGTGACTGTCTAAATCGGTTCATTCGTTGTAACAAATCAGTGTAACAA
    CTCGTGGTACGCCCGGTTTGCTTCGCTCACGCTCCCCTAGATTTTCCGTCTAG
    GACACAACAAGCCCTGGTGGATGACGTTCAACGCCTTCAGCGCGTCTTTTTCC
    TCAATAACACACGAGATGTTAATTTCGTTGGCTCCCTGAGAAATCATCTCAAT
    GTTAATACCTGCCTGGGCCAGAGTAGAGAAGAACATGCCTGCACATCCGACCA
    TGGCCTTCATTCTCGTTCCGATCAGCGACACAATGGTCATGCCTCGCTTCACA
    TCTACTGTGCCGTACTTTCGCAGCTCCTCCACAGCCTGCTTGAGGTTGGAGTC
    GGGAGCATGGAAGGCCATCGACACATGGACCTCAGAAGTGGAGATGAGATCGA
    CGACAAGTTTCTGCTGGTCGAGAGTAGCAAAGATCTTGTTCAAGAAGCCATGA
    CTCTTGGTTCGCTTATTCGAATGAACGTTGAGAACGGTGATATTCGATTTGGT
    AGTGACAGCTGTAGGCTTCTTCTCGTCATCTACAGGAGCAGACACCTGGGGAG
    TGAATGAGCCAGAGGACAGAGAAGTTAGCGAGTCGGTGGAAGAAGCCAGATCC
    TGGACATTGTTTTTCGTTTTCAGGTTGCCCGAGCCGTCAGCAGATGGGTAGAT
    GATAGTTCCTCCTCCCAGGGGGTTTTCCACGTTTTTGATTCGGATAGGAATAT
    GGGCCTTAATGACCTGCTCCATGGTGAAGGGGTGGATGACTTCAGATCCGTAG
    TAGGTCAACTCAGCGGCCTCCTCGGGGGTAATGATGGGCAGCAGACGGGCAGT
    GGACACCTTTCTGGGATCGGCAGTGAAGACACCATCGACTTCTTTCCAGATCT
    GAAGCTCCTTGGCGTCCAGACCCACAGCCACCAGAGCAGCACACAGATCGGTG
    TATCCTCGTCCGATCTGGCTTAGGAGGCCTCCCTTGACGGGGCCAAAGAAACC
    GGTCAACACGGGCACCATGTTTCCCTTGACAGGAGCAGAGTCCGTGTTAGGAG
    AGCCGGGAAGAGTGATGATCTCGCCCAGAACACGTCCGAGGTCCGAATAGAAC
    TTGGGATCTTCGGCATTGGTGGTGGTAACTGCGTGAGACAGGTCAAAGTACCG
    TGCGTTAACACCAGCGTCCCGCATAACTGCAGTCATGTACATGCAACTGAGCT
    TCTCCCCAATGGCCATGATGGAGTCGAGTGTTCGGGGGGAGATCTCAGAAATG
    ATTTCAGCAGCAGCCAGGATTCGCAACAGCTGGTCGCACTCGCCGTTGATGTC
    GGCATTGAGGTTTTCGAGCAGTTCGGGGTTCTTGACGTCACGCTTAGCAGCTG
    CAAGATGGTCTTCTCGGATAGACTCAATGATGGGGTTGTAGGCGTCTGATCCG
    AGAAGAGCAGAGTCAGCAGCAGCAATAAGACGGGTGGTGGTTCCCTCGGCCTT
    GGTGGCCTCTAGATGGCCTCCTTGGCCCCATTCCAGCTGCATTAATGAATCGG
    CCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGC
    TCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCAC
    TCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAA
    CATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGC
    TGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGC
    TCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCC
    CCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGAT
    ACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGC
    TGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCA
    CGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTG
    AGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAAC
    AGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTG
    GCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGA
    AGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACC
    ACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAA
    AAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGT
    GGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATC
    TTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTAT
    ATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTA
    TCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTG
    TAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGAT
    ACCGCGAGAGCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAG
    CCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAG
    TCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTT
    GCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTG
    GTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCC
    CCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAG
    AAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATT
    CTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCA
    ACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGC
    GTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCA
    TTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGA
    TCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTAC
    TTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAA
    AGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAA
    TATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGA
    ATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAG
    TGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAAAAT
    AGGCGTATCACGAG
    MB8388- GGTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCG  9
    LIP8 AGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGG
    ATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGT
    AAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCA
    TCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAA
    GATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACC
    CTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCT
    TTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCA
    AGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCC
    GGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGC
    AGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAG
    AGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGT
    ATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTG
    ATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGC
    AGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACG
    GGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAG
    ATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTA
    AATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTA
    ATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGC
    CTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCC
    CCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCA
    GCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTT
    ATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTT
    CGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTGCAGGCATCGTGGTG
    TCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAG
    GCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTC
    CTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATG
    GCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGT
    GACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGA
    GTTGCTCTTGCCCGGCGTCAACACGGGATAATACCGCGCCACATAGCAGAACT
    TTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGAT
    CTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGAT
    CTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGG
    CAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCAT
    ACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGA
    GCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGC
    ACATTTCCCCGAAAAGTGCCACCTGACGTCTAAGAAACCATTATTATCATGAC
    ATTAACCTATAAAAATAGGCGTATCACGAGGCCCTTTCGTCTGGCCTAGGAAG
    CGACTTCCAATCGCTTTGCATATCCAGTACCACACCCACAGGCGTTTGTGCTA
    CTCTACTGATAGCAATAGATGCGTCATAATTGGTTGGCCCGCTGAGCCTCCAC
    AGGATACTATTGCACATACCCTGGTCATGTGCAGATCAGCTCATTTGTGGAGA
    CTCTGGAGTAACTTAGACGACGCCTGGTTCAATTGCCGCAATGTGCGCCCACG
    CAGATAATGTATTGAGGGGTGGAGCGCCTCTTGGGGACTTGCTGTACTTGTAC
    GGGATATTAAACGCACTCAGCAAGACCATGACGTAAAACACACCTACTGTACG
    ATACGTACTGTAGGTATTGTACTCGTACCCGGTACTACAAATAGTACGATACT
    ATACGGAGTGTATTTGTACCTTGATATACGACTGGCGGAGTGAAGAGAAGGAG
    TTGAACAAGACCAGATGGGGATATCAGCCCCAGTGCTTTGTATTACAAGTACG
    AGTACTTAATAGATACTGTAAGGCTATTGATACGGATGGCAGTAAGTCATTGA
    GTAAGCAATTGTGGCCCAGCATCTCCCCTACGTACTTGTACCATACCCCATGG
    AGACACCAATGGTCTTTCACGCACACTGTCGTGTGCTGTATCGCAGAATCGGG
    TGTCCAACCAAATGCCGTTACCCCCACGTCACAGCCGATAGACAGATACACCA
    TCAATACCAGCAGGTTGTATCATGCGGTTGGCTGAAGGTAAGCTGATTGGTCT
    AAAAACTGTAGCTGTCCTAATTCAACGAGCGCTATTTGGGGCCAACCACCTCG
    GCCAAGCGGCCTTTAATCTGCGTGCCCCAGAGGCGTCTAATGAGGCTCTGGCC
    GCCACTGTAGGAGTGTTTCTCTGTGCGCACACGCAGTTTTGAGTTTGGGCGAC
    TTTCCCTTTTTCCCAATTGCGTACACACACAGCTCCGAGCTAAGCGCTGTCCT
    TGAACCTTCTCCCTCTTTTCCCTCTTTTTCTCTTCCCCTTCCCCTCCTCCACA
    TTAAGGCCAAATCCTGAATTGCACCAACTAGTACAACGACAACAATGGACAAG
    AAGTACTCCATCGGTTTGGACATTGGTACTAACTCTGTCGGCTGGGCCGTCAT
    CACCGACGAGTACAAGGTTCCCTCCAAGAAGTTCAAGGTCCTTGGCAACACCG
    ACCGACACTCTATCAAGAAGAACCTGATCGGTGCTCTGCTGTTCGACTCTGGC
    GAGACTGCCGAGGCCACCCGACTGAAGCGAACCGCTCGACGCCGATACACCCG
    ACGAAAGAACCGAATCTGTTACCTCCAGGAGATCTTCAGCAACGAGATGGCTA
    AGGTCGACGACTCCTTCTTCCACCGACTCGAGGAGTCTTTCCTGGTCGAAGAG
    GATAAGAAGCACGAGCGACACCCCATCTTCGGCAACATTGTTGATGAGGTTGC
    CTACCATGAGAAGTACCCCACCATCTACCACCTCCGAAAGAAGCTCGTCGACT
    CCACTGACAAGGCTGACCTCCGACTCATCTACCTTGCTCTCGCCCACATGATC
    AAGTTCCGAGGTCACTTCCTCATTGAGGGTGATCTCAACCCCGACAACTCCGA
    CGTTGACAAGCTGTTCATCCAGCTCGTCCAGACCTACAACCAGCTCTTTGAGG
    AGAACCCTATCAACGCTTCTGGTGTTGACGCCAAGGCCATTCTCTCCGCCCGA
    CTCTCTAAGTCCCGACGACTCGAGAACCTCATTGCCCAGCTGCCCGGCGAGAA
    GAAGAACGGCCTCTTCGGTAACCTGATTGCTCTCTCTCTTGGTCTGACCCCCA
    ACTTCAAGTCCAACTTTGACCTCGCCGAGGACGCCAAGCTCCAGCTGTCCAAG
    GACACCTACGATGACGATCTGGACAACCTCCTGGCCCAGATCGGTGACCAGTA
    CGCCGATCTCTTCCTTGCCGCCAAGAACCTCTCCGACGCCATCCTGCTCTCCG
    ACATCCTCCGAGTCAACACCGAGATTACCAAGGCTCCTCTGTCTGCCTCTATG
    ATCAAGCGATACGACGAGCACCACCAGGATCTCACTCTTCTCAAGGCTCTCGT
    CCGACAGCAGCTCCCCGAGAAGTACAAGGAGATTTTCTTTGACCAGTCCAAGA
    ACGGTTACGCTGGCTACATTGACGGTGGTGCTTCCCAGGAAGAGTTTTACAAG
    TTCATCAAGCCTATTCTGGAGAAGATGGACGGTACCGAGGAGCTGCTCGTCAA
    GCTCAACCGAGAGGACCTCCTTCGAAAGCAGCGAACCTTCGATAACGGCTCCA
    TCCCCCACCAGATCCACCTGGGTGAGCTCCACGCCATTCTCCGAAGACAAGAG
    GACTTCTACCCCTTCCTAAAGGATAACCGAGAGAAGATCGAGAAGATTCTCAC
    CTTCCGAATCCCCTACTACGTCGGTCCCCTCGCTCGAGGTAACTCCCGATTTG
    CTTGGATGACCCGAAAGTCCGAGGAGACTATCACCCCCTGGAACTTTGAAGAG
    GTAGTCGACAAGGGTGCCTCCGCCCAGTCTTTCATTGAGCGGATGACCAACTT
    CGATAAGAACCTCCCCAACGAGAAGGTCCTTCCCAAGCACTCTCTCCTCTACG
    AGTACTTCACCGTCTACAACGAGCTGACCAAGGTCAAGTACGTTACCGAGGGC
    ATGCGAAAGCCCGCTTTCCTCTCTGGTGAGCAGAAGAAGGCCATTGTCGACCT
    CCTGTTCAAGACTAACCGAAAAGTCACCGTCAAGCAGCTCAAGGAAGACTACT
    TCAAGAAGATTGAGTGCTTCGACTCCGTCGAGATTTCCGGTGTCGAGGACCGA
    TTCAACGCCTCCCTCGGCACCTACCACGATCTTCTGAAGATCATCAAGGACAA
    GGACTTTCTTGATAACGAGGAGAACGAGGACATTCTCGAGGACATCGTCCTCA
    CCCTCACCCTTTTCGAGGATCGAGAGATGATCGAGGAGCGACTCAAGACCTAC
    GCCCATCTCTTCGACGACAAGGTCATGAAGCAACTCAAGCGACGACGATACAC
    TGGCTGGGGCCGACTTTCCCGAAAGCTCATCAACGGCATCCGAGACAAGCAGT
    CTGGCAAGACCATCCTGGACTTCCTGAAGTCCGACGGTTTCGCCAACCGAAAC
    TTCATGCAGCTCATCCACGACGACTCTCTTACCTTCAAAGAGGATATCCAGAA
    GGCCCAGGTTTCTGGCCAGGGCGACTCCCTCCACGAGCACATTGCCAACCTCG
    CCGGATCCCCCGCCATCAAAAAGGGTATCCTCCAGACCGTCAAGGTTGTCGAC
    GAACTCGTGAAGGTCATGGGCCGACACAAGCCCGAGAACATCGTTATCGAGAT
    GGCCCGAGAGAACCAGACCACCCAGAAGGGTCAGAAGAACTCCCGAGAGCGAA
    TGAAGCGAATCGAAGAGGGTATCAAGGAGCTCGGTTCCCAGATTCTCAAGGAG
    CACCCCGTCGAGAACACCCAGCTCCAGAACGAGAAACTCTACCTGTACTACCT
    CCAGAATGGCCGAGACATGTACGTTGACCAGGAGCTCGACATCAACCGACTCT
    CCGACTACGACGTCGACCACATTGTTCCTCAGTCCTTCCTCAAGGACGACTCC
    ATCGACAACAAGGTTCTGACCCGATCTGACAAGAACCGAGGTAAGTCCGACAA
    CGTTCCCTCCGAAGAGGTCGTTAAGAAGATGAAGAACTACTGGCGACAGCTTC
    TCAACGCCAAACTGATCACCCAGCGAAAGTTTGACAACCTCACCAAGGCCGAG
    CGAGGTGGTCTGTCCGAGCTGGACAAGGCCGGCTTCATTAAGCGACAGCTGGT
    CGAGACTCGACAGATCACCAAGCACGTCGCCCAGATCCTCGACTCCCGAATGA
    ACACCAAGTACGACGAGAACGACAAGCTCATCCGGGAGGTCAAGGTCATCACC
    CTGAAGTCTAAGCTTGTCTCCGACTTCCGAAAGGACTTCCAGTTCTACAAGGT
    CCGAGAGATCAACAACTACCACCACGCCCACGACGCCTACCTCAACGCCGTTG
    TTGGTACCGCCCTCATCAAGAAGTATCCCAAGCTCGAGTCCGAGTTCGTTTAC
    GGCGACTACAAGGTTTACGATGTCCGAAAGATGATTGCCAAGTCCGAGCAGGA
    GATCGGTAAGGCCACCGCCAAGTACTTTTTCTACTCCAACATCATGAATTTCT
    TCAAGACCGAGATCACTCTCGCCAACGGTGAGATTCGAAAGCGACCCCTGATT
    GAGACTAATGGTGAGACTGGTGAGATCGTCTGGGATAAGGGCCGAGACTTCGC
    CACCGTCCGAAAGGTCCTGTCCATGCCCCAGGTCAACATTGTCAAGAAGACCG
    AGGTCCAGACCGGTGGCTTCTCCAAGGAGTCCATTCTCCCCAAGCGAAACTCC
    GACAAACTCATCGCCCGTAAGAAGGACTGGGATCCGAAGAAGTACGGTGGTTT
    CGATTCTCCCACCGTTGCCTACTCCGTCCTCGTTGTTGCTAAAGTCGAGAAGG
    GTAAGTCTAAGAAACTCAAGTCCGTGAAGGAGCTACTCGGTATCACCATCATG
    GAGCGATCTTCTTTTGAGAAGAACCCCATTGACTTCCTCGAGGCCAAGGGTTA
    CAAAGAGGTCAAGAAGGACCTGATTATCAAGCTGCCCAAGTACTCCCTCTTTG
    AGCTCGAGAACGGCCGAAAGCGAATGCTGGCTTCCGCTGGTGAGCTGCAGAAG
    GGCAACGAGCTCGCTCTGCCCTCCAAGTACGTCAACTTCCTCTACCTGGCCTC
    CCACTACGAGAAGCTCAAGGGCTCCCCCGAGGACAACGAGCAGAAGCAGCTGT
    TCGTTGAGCAGCACAAGCACTACCTCGACGAGATCATCGAGCAGATCTCCGAG
    TTCTCCAAGCGAGTCATCCTCGCTGACGCCAACCTTGATAAGGTTCTCTCTGC
    TTACAACAAGCACCGGGACAAGCCCATCCGAGAGCAGGCCGAGAATATCATCC
    ACCTCTTCACTCTCACCAACCTCGGCGCTCCTGCTGCCTTCAAGTACTTCGAC
    ACCACCATTGACCGAAAGAGGTACACCTCCACCAAGGAAGTCCTCGACGCCAC
    CCTGATCCACCAGTCCATCACCGGCCTCTACGAAACCCGAATCGACCTCTCCC
    AGCTCGGCGGTGACTCTCGAGCCGACCCCAAGAAGAAGCGAAAAGTCTAAATA
    TCCGAAGATCAAGAGCGAAGCAAGTTGTAAGTCCAGGACATGTTTCCCGCCCA
    CGCGAGTGATTTATAACACCTCTCTTTTTTGACACCCGCTCGCCTTGAAATTC
    ATGTCACATAAATTATAGTCAACGACGTTTGAATAACTTGTCTTGTAGTTCGA
    TGATGATCATATGATTACATTAATAGTAATTACTGTATTTGATATATATACTA
    ATTACAATAGTACATATTAGAACATACAATAGTTAGTGCCGTGAAGTGGCTTA
    AAATACCGCGAGTCGATTACGTAATATTATTACCTCTTGCCCATCGAACGTAC
    AAGTACTCCTCTGTTCTCTCCTTCCTTTGCTTTGTGCACGAAGAACTGCGGTC
    AGGTGACACAACTTTTTCCATCTCAGGGTGTGTCGCGTGTGCTTCATCCAAAC
    TTTAGTTGGGGTTCGGGTTCGCGCGAGATGATCACGTGCCCTGATTTGGTGTC
    GTCCCCCGTCGCGCTGCGCACGTGATTTATTTATTTCCGGTGGCTGCTGTCTA
    CGCGGGGCCTTCTCTGCCCTTCTGTTTCAACCTTCGGGCGGTTCTCGTAACCA
    GCAGTAGCAATCCATTTCGAAACTCAAAGAGCTAAAAACGTTAAACCTCAGCA
    GTCGCTCGACGAATGGGCTGCGGTTGGGAAGCCCACGAGGCCTATAGCCAGAG
    CCTCGAGTTGACAGGAGCCCAGACGCCTTTTCCAACGGCAACTTTTATATAAA
    ATGGCAATGTATTCATGCAATTGCGGCCGTGTCAGGTTGGAGACACTGGACCA
    CACTCTCCATTGCTTCCTGAGGAGATGGATCATTGCTAGTGCATCTACGCGCA
    GCAATCCCGCAAGCTCGACAACCGTAGATGGGCTTTGGTGGGCCAATCAATTA
    CGCAACCCGCACGTTAAATTGTATGAGGAAGGAAGGCCACGGTACAAAGTGGG
    TGGTCTTCACCCAGTGGTTGTTGGTGGCGTCATGCAGACCATGCATTGGGGAT
    AGCACAGGGTTGGGGTGTCTTGTGGACTCAATGGGTGAAAGGAGATGGAAAAG
    GGCGGTGAAAAGTGGTAGAATCGAAATCCCTGACGTCAATTTATAAAGTAAAA
    TGCGTTTCTGCCATTTTGCTCCCCTCCTTCTTTCGCAATCGCCTCCCCAAAAG
    TTGTCGTGGCAGTACACATGCTTGCATACAATGAAGCTAATCCGGCTTGCTCA
    GTAGTTGCTATATCCAGGCATGGTGTGAAACCCCTCAAAGTATATATAGGAGC
    GGTGAGCCCCAGTCTGGGGTCTTTTCTCTCCATCTCAAAACTACTTTCTCACA
    ATGACGAGACTGATGAGTCCGTGAGGACGAAACGAGTAAGCTCGTCGAGGGCT
    GCAAAATTCACGAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTC
    CGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTGGCCGGCATGGTC
    CCAGCCTCCTCGCTGGCGCCGGCTGGGCAACATGCTTCGGCATGGCGAATGGG
    ACTAAACTTCGAGCTAATCCAGTAGCTTACGTTACCCAGGGGCAGGTCAACTG
    GCTAGCCACGAGTCTGTCCCAGGTCGCAATTTAGTGTAATAAACAATATATAT
    ATTGAGTCTAAAGGGAATTGTAGCTATTGTGATTGTGTGATTTTCGTCTTGCT
    GGTTCTTATTGTGTCCCATTCGTTTCATCCTGATGAGGACCCCTGGAACCGGT
    GTTTTCTTAGTCTCTGCAATCGCTAGTCTTGTTGCTATGACAGTTGCGTCGAC
    ACTATTCAGGTCATCTATCGGTTATTCTGATATTATAATACCTCCGGATCGAT
    GTACCTGATTTATACTTGCAGCAATGTTTACTTCTTATCGTTGGACCCCGTCT
    TCAATTACACTTCCCAACTGGGAACACCCCTCTTTATCGACCCATTTTAGGTA
    ATTTACCCTAGCCCATTGTCTCCATAAGGAATATTACCCTAACCCACAGTCCA
    GGGTGCCCAGGTCCTTCTTTGGCCAAATTTTAACTTCGGTCCTATGGCACAGC
    GGTAGCGCGTGAGATTGCAAATCTTAAGGTCCCGAGTTCGAATCTCGGTGGGA
    CCTAGTTGAAAAATACCTCTAATGCGCCGATGGTTTAGTGGTAAAATCCATCG
    TTGCCATCGATGGGCCCCCGGTTCGATTCCGGGTCGGCGCAGGTTGACGTACA
    GCAGGCTGAACGAGGATGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCT
    AGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTTTTTCGC
    GATACACGAATGTGATACGGATCAAAGTAAGCAGGACTACGATAAGATAACGA
    ATGCGGTGCAGTCCATGTCGATTAGGTATAGATACATTTATTTTGTGTTATGT
    TACATTTTGGGGGGATACTGTCCTACTTGTAGTACCTACTTGTAGTGGCGCGT
    CTATTCCTTTGCCCTCGGACGAGTGCTGGGGCGTCGGTTTCCACTATCGGCGA
    GTACTTCTACACAGCCATCGGTCCAGACGGCCGCGCTTCTGCGGGCGATTTGT
    GTACGCCCGACAGTCCCGGCTCCGGATCGGACGATTGCGTCGCATCGACCCTG
    CGCCCAAGCTGCATCATCGAAATTGCCGTCAACCAAGCTCTGATAGAGTTGGT
    CAAGACCAATGCGGAGCATATACGCCCGGAGCCGCGGCGATCCTGCAAGCTCC
    GGATGCCTCCGCTCGAAGTAGCGCGTCTGCTGCTCCATACAAGCCAACCACGG
    CCTCCAGAAGAAGATGTTGGCGACCTCGTATTGGGAATCCCCGAACATCGCCT
    CGCTCCAGTCAATGACCGCTGTTATGCGGCCATTGTCCGTCAGGACATTGTTG
    GAGCCGAAATCCGCGTGCACGAGGTGCCGGACTTCGGGGCAGTCCTCGGCCCA
    AAGCATCAGCTCATCGAGAGCCTGCGCGACGGACGCACTGACGGTGTCGTCCA
    TCACAGTTTGCCAGTGATACACATGGGGATCAGCAATCGCGCATATGAAATCA
    CGCCATGTAGTGTATTGACCGATTCCTTGCGGTCCGAATGGGCCGAACCCGCT
    CGTCTGGCTAAGATCGGCCGCAGCGATCGCATCCATGGCCTCCGCGACCGGCT
    GCAGAACAGCGGGCAGTTCGGTTTCAGGCAGGTCTTGCAACGTGACACCCTGT
    GCACGGCGGGAGATGCAATAGGTCAGGCTCTCGCTGAATTCCCCAATGTCAAG
    CACTTCCGGAATCGGGAGCGCGGCCGATGCAAAGTGCCGATAAACATAACGAT
    CTTTGTAGAAACCATCGGCGCAGCTATTTACCCGCAGGACATATCCACGCCCT
    CCTACATCGAAGCTGAAAGCACGAGATTCTTCGCCCTCCGAGAGCTGCATCAG
    GTCGGAGACGCTGTCGAACTTTTCGATCAGAAACTTCTCGACAGACGTCGCGG
    TGAGTTCAGGCTTTTTCATATGGGTACCTGAGAACATTTTTGTGTCTAGGTGT
    TTGTGTTTGGACTGCGATCAGTGAAGAAAAGAAGAGGAAAAATTGTGCAAGAA
    ATTTTGCTTTCAAGACTTGGCTGATGCAGCAGGGTAACTCTGGGACACAGACC
    TATGTTTGTGGTTAAACTCAATGCACGTGGTACGTGCGTGGAGCGCTTACCCA
    TCCAAGGGTGTGGACATGGAACCGACGGTCCGTGGAGTTGTGTAATGTCATTT
    TGGCGACTCTTGAAGCAAGGCTATAAAAAAATTGTGTGGCTTGAGTCTTATCG
    AGCTCGGTCACTACAAGAGTTAATCTTCCTGTCTCAGGCAGACAGGTCAGGCA
    GGGTTACTTTTGGGTGTGCTGTAACTCACTGTATGGCCGTTAGTGCGCATAGA
    CGTTGTACATACTGGACCGAATTGTAGCGTGCTCAATAGGGCCAATAAAGCTA
    TTGTAGGGATCCGAATTTTCAGAACCTAATTTATCTGTTACCCGGCCTGTGGC
    TCGCACAGCTTAAAAATGGTCAAACTTTCCCCTTCTTGTCTTTTTTTCCTCAC
    ATTCATCAGGTTCTTGTCTTGATCTTTCAAGTGAGTATTAATTACCGACCTTG
    GTTCTTCATTGGGAGAGCATTGGAAGCCGTGGTGCAGCAACCACAAAACGGTT
    CTTCCCCTTCGATACCTTCTTGCCTGCCTTTCAATACAAGTCGGCTCGATTAG
    CGGTGGTCGCCCCCGCCAGCGGAGAACATGGAACTAACCCAGAATGAGAGCTA
    AGTGGAGAAAGAAGAGAGTCAGACGACTCAAGCGAAAGCGCCGCAAGGTCCGA
    GCTCGATCCAAATAAGCGGTTTTTAACGGAGATTTAACACTAAATCGAAGAAC
    TTTTCCCGTTTCATTTGCGAATGAGCTCGTTAACAAAATCCCCCAGTTTTTTT
    ATCCAGCTGTAAGGATTGACATTAGTAATGAATTATTGTTTGGTATATTTAAA
    TCTGTAGTTCCTTTCTGTCCGTGTCGGCAACTGTCGTACTCGTGATTTACTTG
    TATTGACGAATACTTACTGTAGCGCACTCTGCTGCTACTGGTCGTAAGGATGT
    GCTATTTCGGTGTATGGTGGGTTTTTTGGGGGTCGGAACCGAAGACTGTTACA
    CGGGCACGGCTCGTTGTGTACACGCACAGAGCTCTTGCGAGTCATGTTGTAGC
    TAGCTCGTCGTGTTCAGGAACTGTTCGATGGTTCGGAGAGAGTCGCCGCCCAG
    AACATACGCGCACCGATGTCAGCAGACAGCCTTATTACAAGTATATTCAAGCA
    AGTATATCCGTAGGGTGCGGGTGATTTGGATCTAAGGTTCGTACTCAACACTC
    ACGAGCAGCTTGCCTATGTTACATCCTTTTATCAGACATAACATAATTGGAGT
    TTACTTACACACGGGGTGTACCTGTATGAGCACCACCTACAATTGTAGCACTG
    GTACTTGTACAAAGAATTTATTCGTACGAATCACAGGGACGGCCGCCCTCACC
    GAACCAGCGAATACCTCAGCGGTCCCCTGCAGTGACTCAACAAAGCGATATGA
    ACATCTTGCGATGGTATCCTGCTGATAGTTTTTACTGTACAAACACCTGTGTA
    GCTCCTTCTAGCATTTTTAAGTTATTCACACCTCAAGGGGAGGGATAAATTAA
    ATAAATTCCAAAAGCGAAGATCGAGAAACTAAATTAAAATTCCAAAAACGAAG
    TTGGAACACAACCCCCCGAAAAAAAACAACAAACAAAAAACCCAACAAAATAA
    ACAAAAACAAAATAAATATATAACTACCAGTATCTGACTAAAAGTTCAAATAC
    TCGTACTTACAACAAATAGAAATGAGCCGGCCAAAATTCTGCAGAAAAAAATT
    TCAAACAAGTACTGGTATAATTAAATTAAAAAACACATCAAAGTATCATAACG
    TTAGTTATTTTATTTTATTTAATAAAAGAAAACAACAAGATGGGCTCAAAACT
    TTCAACTTATACGATACATACCAAATAACAATTTAGTATTTATCTAAGTGCTT
    TTCGTAGATAATGGAATACAAATGGATATCCAGAGTATACACATGGATAGTAT
    ACACTGACACGACAATTCTGTATCTCTTTATGTTAACTACTGTGAGGCATTAA
    ATAGAGCTTGATATATAAAATGTTACATTTCACAGTCTGAACTTTTGCAGATT
    ACCTAATTTGGTAAGATATTAATTATGAACTGAAAGTTGATGGCATCCCTAAA
    TTTGATGAAAGATGAAATTGTAAATGAGGTGGTAAAAGAGCTACAGTCGTTTT
    GTTTTGAGATACCATCATCTCTAACGAAATATCTATTAAAAATCTCAGTGTGA
    TCATGAGTCATTGCCATCCTGGAAAATGTCATCATGGCTGATATTTCTAACTG
    TTTACTTGAGATAAATATATATTTACAAGAACTTCCCTTGAAATTAATTTAGA
    TATAAAATGTTTGCGGGCAAGTTACTACGAGGAATAAATTATATCTAGAAAGC
    GGAAGAGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATT
    AATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAAC
    GCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTACACTTTAT
    GCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACAG
    GAAACAGCTATGACCATGATTACGCCAAGCTTGGTACCAGAGACGGGTTGGCG
    GCGTATTTGTGTCCCAAAAAACAGCCCCAATTGCCCCAATTGACCCCAAATTG
    ACCCAGTAGCGGGCCCAACCCCGGCGAGAGCCCCCTTCACCCCACATATCAAA
    CCTCCCCCGGTTCCCACACTTGCCGTTAAGGGCGTAGGGTACTGCAGTCTGGA
    ATCTACGCTTGTTCAGACTTTGTACTAGTTTCTTTGTCTGGCCATCCGGGTAA
    CCCATGCCGGACGCAAAATAGACTACTGAAAATTTTTTTGCTTTGTGGTTGGG
    ACTTTAGCCAAGGGTATAAAAGACCACCGTCCCCGAATTACCTTTCCTCTTCT
    TTTCTCTCTCTCCTTGTCAACTCACACCCGAAATCGTTAAGCATTTCCTTCTG
    AGTATAAGAATCATTCGCTAGCCACAAAAATGATGGCTCCCTCCATTCGAAAG
    TTCTTTGCTGGTGGTGTGTGTCGAACTAACGTTCAGCTTCCCGGTAAGGTGGT
    TGTCATCACTGGTGCCAACACCGGCATTGGCAAGGAGACTGCCCGAGAGCTCG
    CTTCCCGAGGAGCCCGAGTTTACATTGCTTGCCGAGATGTTCTGAAGGGCGAG
    TCTGCTGCCTCTGAGATTCGAGTTGACACTAAGAACTCCCAGGTGCTCGTGCG
    AAAGCTCGACCTTTCCGACACTAAGTCTATCCGAGCCTTTGCTGAGGGCTTTC
    TCGCTGAGGAGAAGCAGCTTCACATTCTGATTAACAACGCTGGAGTTATGATG
    TGTCCTTACTCTAAGACTGCTGATGGTTTCGAGACTCACCTCGGAGTCAACCA
    CCTGGGCCACTTCCTCCTCACCTACCTGCTCCTGGAGCGACTCAAGGTGTCTG
    CCCCTGCCCGAGTGGTTAACGTTTCCTCCGTGGCTCACCACATTGGCAAGATT
    CCCTTCCACGACCTCCAGTCCGAGAAGCGATACTCCCGAGGTTTTGCTTACTG
    CCACTCCAAGCTGGCCAACGTTCTCTTTACCCGAGAGCTGGCCAAGCGACTCC
    AGGGAACCGGCGTCACCACCTACGCCGTTCACCCCGGTGTCGTCCGATCCGAG
    CTGGTCCGACACTCCTCCCTGCTCTGCCTGCTCTGGCGACTCTTCTCCCCCTT
    CGTTAAGACCGCCCGAGAGGGTGCCCAGACCTCCCTGCACTGCGCCCTGGCTG
    AGGGCCTGGAGCCCCTGTCTGGCAAGTACTTCTCTGACTGCAAGCGAACCTGG
    GTGTCTCCCCGAGCTCGAAACAACAAGACTGCCGAGCGACTCTGGAACGTTTC
    CTGTGAGCTTCTCGGTATTCGATGGGAGTAGACGCGTGCAATTAACAGATAGT
    TTGCCGGTGATAATTCTCTTAACCTCCCACACTCCTTTGACATAACGATTTAT
    GTAACGAAACTGAAATTTGACCAGATATTGTTGTAAATAGAAAATCTGGCTTG
    TAGGTGGCAAACTAGTGGTGTGTTCTGTGGAGCATTCTCACTTTTGGTAAACG
    ACATTGCTTCAAGTGCAGCGGAATCAAAAAGTATAAAGTGGGCAGCGAGTATA
    CCTGTACAGACTGTAGGCGATAACTCAATCCAATTACCCCCCACAACATGACT
    GGCCAAACTGATCTCAAGACTTTATTGAAATCAGCAACACCGATTCTCAATGA
    AGGCACATACTTCTTCTGCAACATTCACTTGACGCCTAAAGTTGGTGAGAAAT
    GGACCGACAAGACATATTCTGCTATCCACGGACTGTTGCCTGTGTCGGTGGCT
    ACAATACGTGAGTCAGAAGGGCTGACGGTGGTGGTTCCCAAGGAAAAGGTCGA
    CGAGTATCTGTCTGACTCGTCATTGCCGCCTTTGGAGTACGACTCCAACTATG
    AGTGTGCTTGGATCACTTTGACGATACATTCTTCGTTGGAGGCTGTGGGTCTG
    ACAGCTGCGTTTTCGGCGCGGTTGGCCGACAACAATATCAGCTGCAACGTCAT
    TGCTGGCTTTCATCATGATCACATTTTTGTCGGCAAAGGCGACGCCCAGAGAG
    CCATTGACGTTCTTTCTAATTTGGACCGATAGCCGTATAGTCCAGTCTATCTA
    TAAGTTCAACTAACTCGTAACTATTACCATAACATATACTTCACTGCCCCAGA
    TAAGGTTCCGATAAAAAGTTCTGCAGACTAAATTTATTTCAGTCTCCTCTTCA
    CCACCAAAATGCCCTCCTACGAAGCTCGAGCTAACGTCCACAAGTCCGCCTTT
    GCCGCTCGAGTGCTCAAGCTCGTGGCAGCCAAGAAAACCAACCTGTGTGCTTC
    TCTGGATGTTACCACCACCAAGGAGCTCATTGAGCTTGCCGATAAGGTCGGAC
    CTTATGTGTGCATGATCAAGACCCATATCGACATCATTGACGACTTCACCTAC
    GCCGGCACTGTGCTCCCCCTCAAGGAACTTGCTCTTAAGCACGGTTTCTTCCT
    GTTCGAGGACAGAAAGTTCGCAGATATTGGCAACACTGTCAAGCACCAGTACA
    AGAACGGTGTCTACCGAATCGCCGAGTGGTCCGATATCACCAACGCCCACGGT
    GTACCCGGAACCGGAATCATTGCTGGCCTGCGAGCTGGTGCCGAGGAAACTGT
    CTCTGAACAGAAGAAGGAGGACGTCTCTGACTACGAGAACTCCCAGTACAAGG
    AGTTCCTGGTCCCCTCTCCCAACGAGAAGCTGGCCAGAGGTCTGCTCATGCTG
    GCCGAGCTGTCTTGCAAGGGCTCTCTGGCCACTGGCGAGTACTCCAAGCAGAC
    CATTGAGCTTGCCCGATCCGACCCCGAGTTTGTGGTTGGCTTCATTGCCCAGA
    ACCGACCTAAGGGCGACTCTGAGGACTGGCTTATTCTGACCCCCGGGGTGGGT
    CTTGACGACAAGGGAGACGCTCTCGGACAGCAGTACCGAACTGTTGAGGATGT
    CATGTCTACCGGAACGGATATCATAATTGTCGGCCGAGGTCTGTACGGCCAGA
    ACCGAGATCCTATTGAGGAGGCCAAGCGATACCAGAAGGCTGGCTGGGAGGCT
    TACCAGAAGATTAACTGTTAGAGGTTAGACTATGGATATGTAATTTAACTGTG
    TATATAGAGAGCGTGCAAGTATGGAGCGCTTGTTCAGCTTGTATGATGGTCAG
    ACGACCTGTCTGATCGAGTATGTATGATACTGCACAACCTGTGTATCCGCATG
    ATCTGTCCAATGGGGCATGTTGTTGTGTTTCTCGATACGGAGATGCTGGGTAC
    AAGTAGCTAATACGATTGAACTACTTATACTTATATGAGGCTTGAAGAAAGCT
    GACTTGTGTATGACTTATTCTCAACTACATCCCCAGTCACAATACCACCACTG
    CACTACCACTACACCTCGAGCATGCATCTAGTGGTGTGTTCTGTGGAGCATTC
    TCACTTTTGGTAAACGACATTGCTTCAAGTGCAGCGGAATCAAAAAGTATAAA
    GTGGGCAGCGAGTATACCTGTACAGACTGTAGGCGATAACTCAATCCAATTAC
    CCCCCACAACATGACTGGCCAAACTGATCTCAAGACTTTATTGAAATCAGCAA
    CACCGATTCTCAATGAAGGCACATACTTCTTCTGCAACATTCACTTGACGCCT
    AAAGTTGGTGAGAAATGGACCGACAAGACATATTCTGCTATCCACGGACTGTT
    GCCTGTGTCGGTGGCTACAATACGTGAGTCAGAAGGGCTGACGGTGGTGGTTC
    CCAAGGAAAAGGTCGACGAGTATCTGTCTGACTCGTCATTGCCGCCTTTGGAG
    TACGACTCCAACTATGAGTGTGCTTGGATCACTTTGACGATACATTCTTCGTT
    GGAGGCTGTGGGTCTGACAGCTGCGTTTTCGGCGCGGTTGGCCGACAACAATA
    TCAGCTGCAACGTCATTGCTGGCTTTCATCATGATCACATTTTTGTCGGCAAA
    GGCGACGCCCAGAGAGCCATTGACGTTCTTTCTAATTTGGACCGATAGCCGTA
    TAGTCCAGTCTATCTATAAGTTCAACTAACTCGTAACTATTACCATAACATAT
    ACTTCACTGCCCCAGATAAGGTTCCGATAAAAAGTTCTGCAGACTAAATTTAT
    TTCAGTCTCCTCTTCACCACCAAAATGCCCTCCTACGAAGCTCGAGCTAACGT
    CCACAAGTCCGCCTTTGCCGCTCGAGTGCTCAAGCTCGTGGCAGCCAAGAAAA
    CCAACCTGTGTGCTTCTCTGGATGTTACCACCACCAAGGGGCCGCTCGAGCAT
    GCATCTAGAGGGCCCAATTCGCCCTATAGTGAGTCGTATTACAATTCACTGGC
    CGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAATC
    GCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGC
    ACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGGACGCGCCC
    TGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGC
    TACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTC
    TCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTA
    GGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGG
    TGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGA
    CGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACA
    CTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTC
    GGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTA
    ACAAAATTCAGGGCGCAAGGGCTGCTAAAGGAAGCGGAACACGTAGAAAGCCA
    GTCCGCAGAAACGGTGCTGACCCCGGATGAATGTCAGCTACTGGGCTATCTGG
    ACAAGGGAAAACGCAAGCGCAAAGAGAAAGCAGGTAGCTTGCAGTGGGCTTAC
    ATGGCGATAGCTAGACTGGGCGGTTTTATGGACAGCAAGCGAACCGGAATTGC
    CAGCTGGGGCGCCCTCTGGTAAGGTTGGGAAGCCCTGCAAAGTAAACTGGATG
    GCTTTCTTGCCGCCAAGGATCTGATGGCGCAGGGGATCAAGATCTGATCAAGA
    GACAGGATGAGGATCGTTTCGCATGATTGAACAAGATGGATTGCACGCAGGTT
    CTCCGGCCGCTTGGGTGGAGAGGCTATTCGGCTATGACTGGGCACAACAGACA
    ATCGGCTGCTCTGATGCCGCCGTGTTCCGGCTGTCAGCGCAGGGGCGCCCGGT
    TCTTTTTGTCAAGACCGACCTGTCCGGTGCCCTGAATGAACTGCAGGACGAGG
    CAGCGCGGCTATCGTGGCTGGCCACGACGGGCGTTCCTTGCGCAGCTGTGCTC
    GACGTTGTCACTGAAGCGGGAAGGGACTGGCTGCTATTGGGCGAAGTGCCGGG
    GCAGGATCTCCTGTCATCCCACCTTGCTCCTGCCGAGAAAGTATCCATCATGG
    CTGATGCAATGCGGCGGCTGCATACGCTTGATCCGGCTACCTGCCCATTCGAC
    CACCAAGCGAAACATCGCATCGAGCGAGCACGTACTCGGATGGAAGCCGGTCT
    TGTCGATCAGGATGATCTGGACGAAGAGCATCAGGGGCTCGCGCCAGCCGAAC
    TGTTCGCCAGGCTCAAGGCGCGCATGCCCGACGGCGAGGATCTCGTCGTGACC
    CATGGCGATGCCTGCTTGCCGAATATCATGGTGGAAAATGGCCGCTTTTCTGG
    ATTCATCGACTGTGGCCGGCTGGGTGTGGCGGACCGCTATCAGGACATAGCGT
    TGGCTACCCGTGATATTGCTGAAGAGCTTGGCGGCGAATGGGCTGACCGCTTC
    CTCGTGCTTTACGGTATCGCCGCTCCCGATTCGCAGCGCATCGCCTTCTATCG
    CCTTCTTGACGAGTTCTTCTGAATTGAAAAAGGAAGAGTATGAGTATTCAACA
    TTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTG
    CTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCA
    CGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTT
    TCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTG
    GCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATA
    CACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCT
    TACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTG
    ATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTA
    ACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGA
    ACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTG
    TAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTA
    GCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACC
    ACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAG
    CCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAG
    CCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGA
    ACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAAC
    TGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTT
    TAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAAT
    CCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCA
    AAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACA
    AAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAAC
    TCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTTC
    TTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCT
    ACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAA
    GTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGC
    GGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACC
    TACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCC
    CGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAG
    AGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTC
    GGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGG
    GCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCT
    TTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTG
    GATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAAC
    GACCGAGCGCAGCGAGTCAGTGAGCGAGG
    MB8203 AAGCGGAAGAGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATT 10
    CATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCG
    CAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTACACT
    TTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCAC
    ACAGGAAACAGCTATGACCATGATTACGCCAAGCTTGGTACCAGAGACGGGTT
    GGCGGCGTATTTGTGTCCCAAAAAACAGCCCCAATTGCCCCAATTGACCCCAA
    ATTGACCCAGTAGCGGGCCCAACCCCGGCGAGAGCCCCCTTCACCCCACATAT
    CAAACCTCCCCCGGTTCCCACACTTGCCGTTAAGGGCGTAGGGTACTGCAGTC
    TGGAATCTACGCTTGTTCAGACTTTGTACTAGTTTCTTTGTCTGGCCATCCGG
    GTAACCCATGCCGGACGCAAAATAGACTACTGAAAATTTTTTTGCTTTGTGGT
    TGGGACTTTAGCCAAGGGTATAAAAGACCACCGTCCCCGAATTACCTTTCCTC
    TTCTTTTCTCTCTCTCCTTGTCAACTCACACCCGAAATCGTTAAGCATTTCCT
    TCTGAGTATAAGAATCATTCGCTAGCCACAAAAATGATGGCTCCCTCCATTCG
    AAAGTTCTTTGCTGGTGGTGTGTGTCGAACTAACGTTCAGCTTCCCGGTAAGG
    TGGTTGTCATCACTGGTGCCAACACCGGCATTGGCAAGGAGACTGCCCGAGAG
    CTCGCTTCCCGAGGAGCCCGAGTTTACATTGCTTGCCGAGATGTTCTGAAGGG
    CGAGTCTGCTGCCTCTGAGATTCGAGTTGACACTAAGAACTCCCAGGTGCTCG
    TGCGAAAGCTCGACCTTTCCGACACTAAGTCTATCCGAGCCTTTGCTGAGGGC
    TTTCTCGCTGAGGAGAAGCAGCTTCACATTCTGATTAACAACGCTGGAGTTAT
    GATGTGTCCTTACTCTAAGACTGCTGATGGTTTCGAGACTCACCTCGGAGTCA
    ACCACCTGGGCCACTTCCTCCTCACCTACCTGCTCCTGGAGCGACTCAAGGTG
    TCTGCCCCTGCCCGAGTGGTTAACGTTTCCTCCGTGGCTCACCACATTGGCAA
    GATTCCCTTCCACGACCTCCAGTCCGAGAAGCGATACTCCCGAGGTTTTGCTT
    ACTGCCACTCCAAGCTGGCCAACGTTCTCTTTACCCGAGAGCTGGCCAAGCGA
    CTCCAGGGAACCGGCGTCACCACCTACGCCGTTCACCCCGGTGTCGTCCGATC
    CGAGCTGGTCCGACACTCCTCCCTGCTCTGCCTGCTCTGGCGACTCTTCTCCC
    CCTTCGTTAAGACCGCCCGAGAGGGTGCCCAGACCTCCCTGCACTGCGCCCTG
    GCTGAGGGCCTGGAGCCCCTGTCTGGCAAGTACTTCTCTGACTGCAAGCGAAC
    CTGGGTGTCTCCCCGAGCTCGAAACAACAAGACTGCCGAGCGACTCTGGAACG
    TTTCCTGTGAGCTTCTCGGTATTCGATGGGAGTAGACGCGTGCAATTAACAGA
    TAGTTTGCCGGTGATAATTCTCTTAACCTCCCACACTCCTTTGACATAACGAT
    TTATGTAACGAAACTGAAATTTGACCAGATATTGTTGTAAATAGAAAATCTGG
    CTTGTAGGTGGCAAACTAGTGGTGTGTTCTGTGGAGCATTCTCACTTTTGGTA
    AACGACATTGCTTCAAGTGCAGCGGAATCAAAAAGTATAAAGTGGGCAGCGAG
    TATACCTGTACAGACTGTAGGCGATAACTCAATCCAATTACCCCCCACAACAT
    GACTGGCCAAACTGATCTCAAGACTTTATTGAAATCAGCAACACCGATTCTCA
    ATGAAGGCACATACTTCTTCTGCAACATTCACTTGACGCCTAAAGTTGGTGAG
    AAATGGACCGACAAGACATATTCTGCTATCCACGGACTGTTGCCTGTGTCGGT
    GGCTACAATACGTGAGTCAGAAGGGCTGACGGTGGTGGTTCCCAAGGAAAAGG
    TCGACGAGTATCTGTCTGACTCGTCATTGCCGCCTTTGGAGTACGACTCCAAC
    TATGAGTGTGCTTGGATCACTTTGACGATACATTCTTCGTTGGAGGCTGTGGG
    TCTGACAGCTGCGTTTTCGGCGCGGTTGGCCGACAACAATATCAGCTGCAACG
    TCATTGCTGGCTTTCATCATGATCACATTTTTGTCGGCAAAGGCGACGCCCAG
    AGAGCCATTGACGTTCTTTCTAATTTGGACCGATAGCCGTATAGTCCAGTCTA
    TCTATAAGTTCAACTAACTCGTAACTATTACCATAACATATACTTCACTGCCC
    CAGATAAGGTTCCGATAAAAAGTTCTGCAGACTAAATTTATTTCAGTCTCCTC
    TTCACCACCAAAATGCCCTCCTACGAAGCTCGAGCTAACGTCCACAAGTCCGC
    CTTTGCCGCTCGAGTGCTCAAGCTCGTGGCAGCCAAGAAAACCAACCTGTGTG
    CTTCTCTGGATGTTACCACCACCAAGGAGCTCATTGAGCTTGCCGATAAGGTC
    GGACCTTATGTGTGCATGATCAAGACCCATATCGACATCATTGACGACTTCAC
    CTACGCCGGCACTGTGCTCCCCCTCAAGGAACTTGCTCTTAAGCACGGTTTCT
    TCCTGTTCGAGGACAGAAAGTTCGCAGATATTGGCAACACTGTCAAGCACCAG
    TACAAGAACGGTGTCTACCGAATCGCCGAGTGGTCCGATATCACCAACGCCCA
    CGGTGTACCCGGAACCGGAATCATTGCTGGCCTGCGAGCTGGTGCCGAGGAAA
    CTGTCTCTGAACAGAAGAAGGAGGACGTCTCTGACTACGAGAACTCCCAGTAC
    AAGGAGTTCCTGGTCCCCTCTCCCAACGAGAAGCTGGCCAGAGGTCTGCTCAT
    GCTGGCCGAGCTGTCTTGCAAGGGCTCTCTGGCCACTGGCGAGTACTCCAAGC
    AGACCATTGAGCTTGCCCGATCCGACCCCGAGTTTGTGGTTGGCTTCATTGCC
    CAGAACCGACCTAAGGGCGACTCTGAGGACTGGCTTATTCTGACCCCCGGGGT
    GGGTCTTGACGACAAGGGAGACGCTCTCGGACAGCAGTACCGAACTGTTGAGG
    ATGTCATGTCTACCGGAACGGATATCATAATTGTCGGCCGAGGTCTGTACGGC
    CAGAACCGAGATCCTATTGAGGAGGCCAAGCGATACCAGAAGGCTGGCTGGGA
    GGCTTACCAGAAGATTAACTGTTAGAGGTTAGACTATGGATATGTAATTTAAC
    TGTGTATATAGAGAGCGTGCAAGTATGGAGCGCTTGTTCAGCTTGTATGATGG
    TCAGACGACCTGTCTGATCGAGTATGTATGATACTGCACAACCTGTGTATCCG
    CATGATCTGTCCAATGGGGCATGTTGTTGTGTTTCTCGATACGGAGATGCTGG
    GTACAAGTAGCTAATACGATTGAACTACTTATACTTATATGAGGCTTGAAGAA
    AGCTGACTTGTGTATGACTTATTCTCAACTACATCCCCAGTCACAATACCACC
    ACTGCACTACCACTACACCTCGAGCATGCATCTAGTGGTGTGTTCTGTGGAGC
    ATTCTCACTTTTGGTAAACGACATTGCTTCAAGTGCAGCGGAATCAAAAAGTA
    TAAAGTGGGCAGCGAGTATACCTGTACAGACTGTAGGCGATAACTCAATCCAA
    TTACCCCCCACAACATGACTGGCCAAACTGATCTCAAGACTTTATTGAAATCA
    GCAACACCGATTCTCAATGAAGGCACATACTTCTTCTGCAACATTCACTTGAC
    GCCTAAAGTTGGTGAGAAATGGACCGACAAGACATATTCTGCTATCCACGGAC
    TGTTGCCTGTGTCGGTGGCTACAATACGTGAGTCAGAAGGGCTGACGGTGGTG
    GTTCCCAAGGAAAAGGTCGACGAGTATCTGTCTGACTCGTCATTGCCGCCTTT
    GGAGTACGACTCCAACTATGAGTGTGCTTGGATCACTTTGACGATACATTCTT
    CGTTGGAGGCTGTGGGTCTGACAGCTGCGTTTTCGGCGCGGTTGGCCGACAAC
    AATATCAGCTGCAACGTCATTGCTGGCTTTCATCATGATCACATTTTTGTCGG
    CAAAGGCGACGCCCAGAGAGCCATTGACGTTCTTTCTAATTTGGACCGATAGC
    CGTATAGTCCAGTCTATCTATAAGTTCAACTAACTCGTAACTATTACCATAAC
    ATATACTTCACTGCCCCAGATAAGGTTCCGATAAAAAGTTCTGCAGACTAAAT
    TTATTTCAGTCTCCTCTTCACCACCAAAATGCCCTCCTACGAAGCTCGAGCTA
    ACGTCCACAAGTCCGCCTTTGCCGCTCGAGTGCTCAAGCTCGTGGCAGCCAAG
    AAAACCAACCTGTGTGCTTCTCTGGATGTTACCACCACCAAGGGGCCGCTCGA
    GCATGCATCTAGAGGGCCCAATTCGCCCTATAGTGAGTCGTATTACAATTCAC
    TGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTT
    AATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGC
    CCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGGACGC
    GCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGA
    CCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCC
    TTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCC
    TTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATT
    AGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCT
    TTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAAC
    AACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGA
    TTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAAT
    TTTAACAAAATTCAGGGCGCAAGGGCTGCTAAAGGAAGCGGAACACGTAGAAA
    GCCAGTCCGCAGAAACGGTGCTGACCCCGGATGAATGTCAGCTACTGGGCTAT
    CTGGACAAGGGAAAACGCAAGCGCAAAGAGAAAGCAGGTAGCTTGCAGTGGGC
    TTACATGGCGATAGCTAGACTGGGCGGTTTTATGGACAGCAAGCGAACCGGAA
    TTGCCAGCTGGGGCGCCCTCTGGTAAGGTTGGGAAGCCCTGCAAAGTAAACTG
    GATGGCTTTCTTGCCGCCAAGGATCTGATGGCGCAGGGGATCAAGATCTGATC
    AAGAGACAGGATGAGGATCGTTTCGCATGATTGAACAAGATGGATTGCACGCA
    GGTTCTCCGGCCGCTTGGGTGGAGAGGCTATTCGGCTATGACTGGGCACAACA
    GACAATCGGCTGCTCTGATGCCGCCGTGTTCCGGCTGTCAGCGCAGGGGCGCC
    CGGTTCTTTTTGTCAAGACCGACCTGTCCGGTGCCCTGAATGAACTGCAGGAC
    GAGGCAGCGCGGCTATCGTGGCTGGCCACGACGGGCGTTCCTTGCGCAGCTGT
    GCTCGACGTTGTCACTGAAGCGGGAAGGGACTGGCTGCTATTGGGCGAAGTGC
    CGGGGCAGGATCTCCTGTCATCCCACCTTGCTCCTGCCGAGAAAGTATCCATC
    ATGGCTGATGCAATGCGGCGGCTGCATACGCTTGATCCGGCTACCTGCCCATT
    CGACCACCAAGCGAAACATCGCATCGAGCGAGCACGTACTCGGATGGAAGCCG
    GTCTTGTCGATCAGGATGATCTGGACGAAGAGCATCAGGGGCTCGCGCCAGCC
    GAACTGTTCGCCAGGCTCAAGGCGCGCATGCCCGACGGCGAGGATCTCGTCGT
    GACCCATGGCGATGCCTGCTTGCCGAATATCATGGTGGAAAATGGCCGCTTTT
    CTGGATTCATCGACTGTGGCCGGCTGGGTGTGGCGGACCGCTATCAGGACATA
    GCGTTGGCTACCCGTGATATTGCTGAAGAGCTTGGCGGCGAATGGGCTGACCG
    CTTCCTCGTGCTTTACGGTATCGCCGCTCCCGATTCGCAGCGCATCGCCTTCT
    ATCGCCTTCTTGACGAGTTCTTCTGAATTGAAAAAGGAAGAGTATGAGTATTC
    AACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTT
    TTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGG
    TGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGA
    GTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTA
    TGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCG
    CATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGC
    ATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATG
    AGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGA
    GCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTT
    GGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATG
    CCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTAC
    TCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAG
    GACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCT
    GGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGG
    TAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGG
    ATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGG
    TAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCA
    TTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCA
    AAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAG
    ATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCA
    AACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTAC
    CAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACT
    GTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACC
    GCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCG
    ATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCG
    CAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAAC
    GACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGC
    TTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACA
    GGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCC
    TGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAG
    GGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTG
    GCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTC
    TGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCC
    GAACGACCGAGCGCAGCGAGTCAGTGAGCGAGG
    MB9894 AAGCGGAAGAGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATT 11
    CATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCG
    CAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTACACT
    TTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCAC
    ACAGGAAACAGCTATGACCATGATTACGCCAAGCTTGGTACCAGAGACGGGTT
    GGCGGCGTATTTGTGTCCCAAAAAACAGCCCCAATTGCCCCAATTGACCCCAA
    ATTGACCCAGTAGCGGGCCCAACCCCGGCGAGAGCCCCCTTCACCCCACATAT
    CAAACCTCCCCCGGTTCCCACACTTGCCGTTAAGGGCGTAGGGTACTGCAGTC
    TGGAATCTACGCTTGTTCAGACTTTGTACTAGTTTCTTTGTCTGGCCATCCGG
    GTAACCCATGCCGGACGCAAAATAGACTACTGAAAATTTTTTTGCTTTGTGGT
    TGGGACTTTAGCCAAGGGTATAAAAGACCACCGTCCCCGAATTACCTTTCCTC
    TTCTTTTCTCTCTCTCCTTGTCAACTCACACCCGAAATCGTTAAGCATTTCCT
    TCTGAGTATAAGAATCATTCGCTAGCCCACAAAAATGACCCGATCCAAGTTCA
    CCGAGTACAAGCAGGTTCTCCGAAACTCTTGTGTTGGCAAGCCCCAGTACACT
    GAGAAGGATTACCCCGATCTCGACGGAAAGAACTTTCTCGTCACTGGTGCTAC
    TGGTGGAGTTGGTCTGGAGGCCACTAAGCTGCTCCTTGAGAAGAAGTCTCACG
    TTATTATGGTTGGCCGATCTAAGACTAAGTCTCAGTCTACCCTTGACGAGCTT
    CAGAAGACTTACTCTCACGGTACTTTCGATTTCGTTGAGGCTGATCTCTCTGA
    CCTTACCACTGTTGAGCGAGCCGGAGAGTACATTCGATCTAAGTACACTACCC
    TTGACGGTGCCATTCTTAACGCTGGAGTCATGGCTCCCCCCTACTCTCTCACT
    CCCCAGGGTCACGAGTCTCAGTGGGGTATTAACGTTGTTGCTCACTTTCTCCT
    GTCTAAGTACATCTCTCCTGCTCTGATTTCTGCTGCTCAGACTGCCCCCAAGG
    ACACCGTTCGACTTGTTTGGGTGTCTTCTTCTGTCGTTGCCATGTCTCCTTAC
    GAGGGAGGTATTAAGTTTGACGATATTAACCACTCTAAGGTCAAGAACCCTTC
    TCCTTGGACCCTTTACTCTCAGTCTAAGATTGGTGATGCTTACCTGGCTTACC
    TTTGGTCTAAGCACCACCCCGATTCTGGAGTCCTTTCTGTCTCTCTGGACCCC
    GGTAACCTGGCTTCTGATCTCTCTCGACACACTTCTTGGCTTTCTTCTATTAA
    GAACTACGTTCTTTACCCTCCCAAGTACGGTGCCTACACTGAGCTGTCTGCTC
    TTCTCAACCCTTCCGTTAAGAACAACGAGCACCTTATTCCCTGGGGTATTGAG
    GGACACCTTCGACAGGATGTCGACGATGGTCGACGAGGAAAGGACGGAGAGGA
    GCTCTGGCAGGGTCTTAACAAGGATGTCGAGGGTTTCTTTAAGGAGGAGTAAA
    CGCGTGCAATTAACAGATAGTTTGCCGGTGATAATTCTCTTAACCTCCCACAC
    TCCTTTGACATAACGATTTATGTAACGAAACTGAAATTTGACCAGATATTGTT
    GTAAATAGAAAATCTGGCTTGTAGGTGGCAAACTAGTGGTGTGTTCTGTGGAG
    CATTCTCACTTTTGGTAAACGACATTGCTTCAAGTGCAGCGGAATCAAAAAGT
    ATAAAGTGGGCAGCGAGTATACCTGTACAGACTGTAGGCGATAACTCAATCCA
    ATTACCCCCCACAACATGACTGGCCAAACTGATCTCAAGACTTTATTGAAATC
    AGCAACACCGATTCTCAATGAAGGCACATACTTCTTCTGCAACATTCACTTGA
    CGCCTAAAGTTGGTGAGAAATGGACCGACAAGACATATTCTGCTATCCACGGA
    CTGTTGCCTGTGTCGGTGGCTACAATACGTGAGTCAGAAGGGCTGACGGTGGT
    GGTTCCCAAGGAAAAGGTCGACGAGTATCTGTCTGACTCGTCATTGCCGCCTT
    TGGAGTACGACTCCAACTATGAGTGTGCTTGGATCACTTTGACGATACATTCT
    TCGTTGGAGGCTGTGGGTCTGACAGCTGCGTTTTCGGCGCGGTTGGCCGACAA
    CAATATCAGCTGCAACGTCATTGCTGGCTTTCATCATGATCACATTTTTGTCG
    GCAAAGGCGACGCCCAGAGAGCCATTGACGTTCTTTCTAATTTGGACCGATAG
    CCGTATAGTCCAGTCTATCTATAAGTTCAACTAACTCGTAACTATTACCATAA
    CATATACTTCACTGCCCCAGATAAGGTTCCGATAAAAAGTTCTGCAGACTAAA
    TTTATTTCAGTCTCCTCTTCACCACCAAAATGCCCTCCTACGAAGCTCGAGCT
    AACGTCCACAAGTCCGCCTTTGCCGCTCGAGTGCTCAAGCTCGTGGCAGCCAA
    GAAAACCAACCTGTGTGCTTCTCTGGATGTTACCACCACCAAGGAGCTCATTG
    AGCTTGCCGATAAGGTCGGACCTTATGTGTGCATGATCAAGACCCATATCGAC
    ATCATTGACGACTTCACCTACGCCGGCACTGTGCTCCCCCTCAAGGAACTTGC
    TCTTAAGCACGGTTTCTTCCTGTTCGAGGACAGAAAGTTCGCAGATATTGGCA
    ACACTGTCAAGCACCAGTACAAGAACGGTGTCTACCGAATCGCCGAGTGGTCC
    GATATCACCAACGCCCACGGTGTACCCGGAACCGGAATCATTGCTGGCCTGCG
    AGCTGGTGCCGAGGAAACTGTCTCTGAACAGAAGAAGGAGGACGTCTCTGACT
    ACGAGAACTCCCAGTACAAGGAGTTCCTGGTCCCCTCTCCCAACGAGAAGCTG
    GCCAGAGGTCTGCTCATGCTGGCCGAGCTGTCTTGCAAGGGCTCTCTGGCCAC
    TGGCGAGTACTCCAAGCAGACCATTGAGCTTGCCCGATCCGACCCCGAGTTTG
    TGGTTGGCTTCATTGCCCAGAACCGACCTAAGGGCGACTCTGAGGACTGGCTT
    ATTCTGACCCCCGGGGTGGGTCTTGACGACAAGGGAGACGCTCTCGGACAGCA
    GTACCGAACTGTTGAGGATGTCATGTCTACCGGAACGGATATCATAATTGTCG
    GCCGAGGTCTGTACGGCCAGAACCGAGATCCTATTGAGGAGGCCAAGCGATAC
    CAGAAGGCTGGCTGGGAGGCTTACCAGAAGATTAACTGTTAGAGGTTAGACTA
    TGGATATGTAATTTAACTGTGTATATAGAGAGCGTGCAAGTATGGAGCGCTTG
    TTCAGCTTGTATGATGGTCAGACGACCTGTCTGATCGAGTATGTATGATACTG
    CACAACCTGTGTATCCGCATGATCTGTCCAATGGGGCATGTTGTTGTGTTTCT
    CGATACGGAGATGCTGGGTACAAGTAGCTAATACGATTGAACTACTTATACTT
    ATATGAGGCTTGAAGAAAGCTGACTTGTGTATGACTTATTCTCAACTACATCC
    CCAGTCACAATACCACCACTGCACTACCACTACACCTCGAGCATGCATCTAGT
    GGTGTGTTCTGTGGAGCATTCTCACTTTTGGTAAACGACATTGCTTCAAGTGC
    AGCGGAATCAAAAAGTATAAAGTGGGCAGCGAGTATACCTGTACAGACTGTAG
    GCGATAACTCAATCCAATTACCCCCCACAACATGACTGGCCAAACTGATCTCA
    AGACTTTATTGAAATCAGCAACACCGATTCTCAATGAAGGCACATACTTCTTC
    TGCAACATTCACTTGACGCCTAAAGTTGGTGAGAAATGGACCGACAAGACATA
    TTCTGCTATCCACGGACTGTTGCCTGTGTCGGTGGCTACAATACGTGAGTCAG
    AAGGGCTGACGGTGGTGGTTCCCAAGGAAAAGGTCGACGAGTATCTGTCTGAC
    TCGTCATTGCCGCCTTTGGAGTACGACTCCAACTATGAGTGTGCTTGGATCAC
    TTTGACGATACATTCTTCGTTGGAGGCTGTGGGTCTGACAGCTGCGTTTTCGG
    CGCGGTTGGCCGACAACAATATCAGCTGCAACGTCATTGCTGGCTTTCATCAT
    GATCACATTTTTGTCGGCAAAGGCGACGCCCAGAGAGCCATTGACGTTCTTTC
    TAATTTGGACCGATAGCCGTATAGTCCAGTCTATCTATAAGTTCAACTAACTC
    GTAACTATTACCATAACATATACTTCACTGCCCCAGATAAGGTTCCGATAAAA
    AGTTCTGCAGACTAAATTTATTTCAGTCTCCTCTTCACCACCAAAATGCCCTC
    CTACGAAGCTCGAGCTAACGTCCACAAGTCCGCCTTTGCCGCTCGAGTGCTCA
    AGCTCGTGGCAGCCAAGAAAACCAACCTGTGTGCTTCTCTGGATGTTACCACC
    ACCAAGGGGCCGCTCGAGCATGCATCTAGAGGGCCCAATTCGCCCTATAGTGA
    GTCGTATTACAATTCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACC
    CTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGG
    CGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCT
    GAATGGCGAATGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGT
    GGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTT
    TCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCT
    CTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGA
    CCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGAT
    AGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTC
    TTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTT
    ATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAAC
    AAAAATTTAACGCGAATTTTAACAAAATTCAGGGCGCAAGGGCTGCTAAAGGA
    AGCGGAACACGTAGAAAGCCAGTCCGCAGAAACGGTGCTGACCCCGGATGAAT
    GTCAGCTACTGGGCTATCTGGACAAGGGAAAACGCAAGCGCAAAGAGAAAGCA
    GGTAGCTTGCAGTGGGCTTACATGGCGATAGCTAGACTGGGCGGTTTTATGGA
    CAGCAAGCGAACCGGAATTGCCAGCTGGGGCGCCCTCTGGTAAGGTTGGGAAG
    CCCTGCAAAGTAAACTGGATGGCTTTCTTGCCGCCAAGGATCTGATGGCGCAG
    GGGATCAAGATCTGATCAAGAGACAGGATGAGGATCGTTTCGCATGATTGAAC
    AAGATGGATTGCACGCAGGTTCTCCGGCCGCTTGGGTGGAGAGGCTATTCGGC
    TATGACTGGGCACAACAGACAATCGGCTGCTCTGATGCCGCCGTGTTCCGGCT
    GTCAGCGCAGGGGCGCCCGGTTCTTTTTGTCAAGACCGACCTGTCCGGTGCCC
    TGAATGAACTGCAGGACGAGGCAGCGCGGCTATCGTGGCTGGCCACGACGGGC
    GTTCCTTGCGCAGCTGTGCTCGACGTTGTCACTGAAGCGGGAAGGGACTGGCT
    GCTATTGGGCGAAGTGCCGGGGCAGGATCTCCTGTCATCCCACCTTGCTCCTG
    CCGAGAAAGTATCCATCATGGCTGATGCAATGCGGCGGCTGCATACGCTTGAT
    CCGGCTACCTGCCCATTCGACCACCAAGCGAAACATCGCATCGAGCGAGCACG
    TACTCGGATGGAAGCCGGTCTTGTCGATCAGGATGATCTGGACGAAGAGCATC
    AGGGGCTCGCGCCAGCCGAACTGTTCGCCAGGCTCAAGGCGCGCATGCCCGAC
    GGCGAGGATCTCGTCGTGACCCATGGCGATGCCTGCTTGCCGAATATCATGGT
    GGAAAATGGCCGCTTTTCTGGATTCATCGACTGTGGCCGGCTGGGTGTGGCGG
    ACCGCTATCAGGACATAGCGTTGGCTACCCGTGATATTGCTGAAGAGCTTGGC
    GGCGAATGGGCTGACCGCTTCCTCGTGCTTTACGGTATCGCCGCTCCCGATTC
    GCAGCGCATCGCCTTCTATCGCCTTCTTGACGAGTTCTTCTGAATTGAAAAAG
    GAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGG
    CATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGAT
    GCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAG
    CGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCA
    CTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAA
    GAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTC
    ACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCA
    GTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACG
    ATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGT
    AACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACG
    AGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTA
    ACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGA
    GGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGT
    TTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCA
    GCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGG
    GAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCT
    CACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAG
    ATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTT
    TGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGT
    CAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGC
    GTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTT
    GCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAG
    CGCAGATACCAAATACTGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTC
    AAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGT
    GGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGAT
    AGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAG
    CCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCT
    ATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAA
    GCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCC
    TGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATT
    TTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGG
    CCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCT
    GCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGA
    TACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGG
    MB9997 AAGCGGAAGAGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATT 12
    CATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCG
    CAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTACACT
    TTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCAC
    ACAGGAAACAGCTATGACCATGATTACGCCAAGCTTGGTACCAGAGACGGGTT
    GGCGGCGTATTTGTGTCCCAAAAAACAGCCCCAATTGCCCCAATTGACCCCAA
    ATTGACCCAGTAGCGGGCCCAACCCCGGCGAGAGCCCCCTTCACCCCACATAT
    CAAACCTCCCCCGGTTCCCACACTTGCCGTTAAGGGCGTAGGGTACTGCAGTC
    TGGAATCTACGCTTGTTCAGACTTTGTACTAGTTTCTTTGTCTGGCCATCCGG
    GTAACCCATGCCGGACGCAAAATAGACTACTGAAAATTTTTTTGCTTTGTGGT
    TGGGACTTTAGCCAAGGGTATAAAAGACCACCGTCCCCGAATTACCTTTCCTC
    TTCTTTTCTCTCTCTCCTTGTCAACTCACACCCGAAATCGTTAAGCATTTCCT
    TCTGAGTATAAGAATCATTCGCTAGCCCACAAAAATGGTTAACGTTGCTTACG
    AGTGGCTTCGAGACTGCTGGCCCCCTAAGCCTGAGTTCCTCCCTAAGGATTAC
    CCTGACCTCACCGGAAAGACCATCCTTATCACTGGTGCCAACACTGGTATTGG
    TTTTGAGTCCGCTAAGGCTCTCCTCAAGAAGAACGCTACCGTTGTTTTTGCCA
    ACCGATCCGAGGAGAAGACTAAGATCGCTATTGCCAAGATTCAGGCTGAGCTT
    GGTGGTGATTCTTCTCAGCGATCTATTTTCGTCAAGACTGACCTTTCCGATCT
    CTCTTCCATTAAGGGCACTGCTGAGGAGCTTAAGTCTAAGGGTATCACTAAGC
    TCCACTACACCATTCTTAACGCTGGTGTTATGCAGCCTCCCAAGGGCTCCAAG
    ACTAAGCAGGGTTTTGAGCTTCAGATTGGTACTAACGTTCTTGGCCACCAGCT
    CCTCCAGAAGTTTCTCACTCCCCTTGTTCTCAACGCTGTTTCTTCTGATTTCA
    CCCCTCGAGTTGTTTGGCTCGCCTCTGCTGCTCACCTTTCTTCCCCTCCCAAC
    GGTGGTATTGATTGGGATTCCTTCCGAAACGCTGATTGGGCTGGTACTGTCTC
    TGCTTACGGTCAGTCTAAGACTGGTAACATTTACCAGGCTTACATCTACGCTC
    AGCAGCACAAGGATGTCATCTCTGTCTCTGCTCACCCCGGATACCTTGCTTCT
    GATCTCACTCGAGCTTACAAGGGATACCTTCAGTACCTTATGAAGCTCCTTAT
    GTCTCCTCCCGTTTACGGTTCTTACACTGAGCTTTTCGCTGCTCTTTCTCCCA
    AGGTTACTCTCAAGGAGTCTGGTCGATACATTGGTCCTTGGGGTCAGTTCCGA
    GAGCTTCGAGATGACGTTCAGGAGGGCCTTACCGATGGTACTGCTCAGAAGCT
    CTGGGATTGGGCTGAGGCTGAGATTCGACCTTACACTTAAACGCGTGCAATTA
    ACAGATAGTTTGCCGGTGATAATTCTCTTAACCTCCCACACTCCTTTGACATA
    ACGATTTATGTAACGAAACTGAAATTTGACCAGATATTGTTGTAAATAGAAAA
    TCTGGCTTGTAGGTGGCAAACTAGTGGTGTGTTCTGTGGAGCATTCTCACTTT
    TGGTAAACGACATTGCTTCAAGTGCAGCGGAATCAAAAAGTATAAAGTGGGCA
    GCGAGTATACCTGTACAGACTGTAGGCGATAACTCAATCCAATTACCCCCCAC
    AACATGACTGGCCAAACTGATCTCAAGACTTTATTGAAATCAGCAACACCGAT
    TCTCAATGAAGGCACATACTTCTTCTGCAACATTCACTTGACGCCTAAAGTTG
    GTGAGAAATGGACCGACAAGACATATTCTGCTATCCACGGACTGTTGCCTGTG
    TCGGTGGCTACAATACGTGAGTCAGAAGGGCTGACGGTGGTGGTTCCCAAGGA
    AAAGGTCGACGAGTATCTGTCTGACTCGTCATTGCCGCCTTTGGAGTACGACT
    CCAACTATGAGTGTGCTTGGATCACTTTGACGATACATTCTTCGTTGGAGGCT
    GTGGGTCTGACAGCTGCGTTTTCGGCGCGGTTGGCCGACAACAATATCAGCTG
    CAACGTCATTGCTGGCTTTCATCATGATCACATTTTTGTCGGCAAAGGCGACG
    CCCAGAGAGCCATTGACGTTCTTTCTAATTTGGACCGATAGCCGTATAGTCCA
    GTCTATCTATAAGTTCAACTAACTCGTAACTATTACCATAACATATACTTCAC
    TGCCCCAGATAAGGTTCCGATAAAAAGTTCTGCAGACTAAATTTATTTCAGTC
    TCCTCTTCACCACCAAAATGCCCTCCTACGAAGCTCGAGCTAACGTCCACAAG
    TCCGCCTTTGCCGCTCGAGTGCTCAAGCTCGTGGCAGCCAAGAAAACCAACCT
    GTGTGCTTCTCTGGATGTTACCACCACCAAGGAGCTCATTGAGCTTGCCGATA
    AGGTCGGACCTTATGTGTGCATGATCAAGACCCATATCGACATCATTGACGAC
    TTCACCTACGCCGGCACTGTGCTCCCCCTCAAGGAACTTGCTCTTAAGCACGG
    TTTCTTCCTGTTCGAGGACAGAAAGTTCGCAGATATTGGCAACACTGTCAAGC
    ACCAGTACAAGAACGGTGTCTACCGAATCGCCGAGTGGTCCGATATCACCAAC
    GCCCACGGTGTACCCGGAACCGGAATCATTGCTGGCCTGCGAGCTGGTGCCGA
    GGAAACTGTCTCTGAACAGAAGAAGGAGGACGTCTCTGACTACGAGAACTCCC
    AGTACAAGGAGTTCCTGGTCCCCTCTCCCAACGAGAAGCTGGCCAGAGGTCTG
    CTCATGCTGGCCGAGCTGTCTTGCAAGGGCTCTCTGGCCACTGGCGAGTACTC
    CAAGCAGACCATTGAGCTTGCCCGATCCGACCCCGAGTTTGTGGTTGGCTTCA
    TTGCCCAGAACCGACCTAAGGGCGACTCTGAGGACTGGCTTATTCTGACCCCC
    GGGGTGGGTCTTGACGACAAGGGAGACGCTCTCGGACAGCAGTACCGAACTGT
    TGAGGATGTCATGTCTACCGGAACGGATATCATAATTGTCGGCCGAGGTCTGT
    ACGGCCAGAACCGAGATCCTATTGAGGAGGCCAAGCGATACCAGAAGGCTGGC
    TGGGAGGCTTACCAGAAGATTAACTGTTAGAGGTTAGACTATGGATATGTAAT
    TTAACTGTGTATATAGAGAGCGTGCAAGTATGGAGCGCTTGTTCAGCTTGTAT
    GATGGTCAGACGACCTGTCTGATCGAGTATGTATGATACTGCACAACCTGTGT
    ATCCGCATGATCTGTCCAATGGGGCATGTTGTTGTGTTTCTCGATACGGAGAT
    GCTGGGTACAAGTAGCTAATACGATTGAACTACTTATACTTATATGAGGCTTG
    AAGAAAGCTGACTTGTGTATGACTTATTCTCAACTACATCCCCAGTCACAATA
    CCACCACTGCACTACCACTACACCTCGAGCATGCATCTAGTGGTGTGTTCTGT
    GGAGCATTCTCACTTTTGGTAAACGACATTGCTTCAAGTGCAGCGGAATCAAA
    AAGTATAAAGTGGGCAGCGAGTATACCTGTACAGACTGTAGGCGATAACTCAA
    TCCAATTACCCCCCACAACATGACTGGCCAAACTGATCTCAAGACTTTATTGA
    AATCAGCAACACCGATTCTCAATGAAGGCACATACTTCTTCTGCAACATTCAC
    TTGACGCCTAAAGTTGGTGAGAAATGGACCGACAAGACATATTCTGCTATCCA
    CGGACTGTTGCCTGTGTCGGTGGCTACAATACGTGAGTCAGAAGGGCTGACGG
    TGGTGGTTCCCAAGGAAAAGGTCGACGAGTATCTGTCTGACTCGTCATTGCCG
    CCTTTGGAGTACGACTCCAACTATGAGTGTGCTTGGATCACTTTGACGATACA
    TTCTTCGTTGGAGGCTGTGGGTCTGACAGCTGCGTTTTCGGCGCGGTTGGCCG
    ACAACAATATCAGCTGCAACGTCATTGCTGGCTTTCATCATGATCACATTTTT
    GTCGGCAAAGGCGACGCCCAGAGAGCCATTGACGTTCTTTCTAATTTGGACCG
    ATAGCCGTATAGTCCAGTCTATCTATAAGTTCAACTAACTCGTAACTATTACC
    ATAACATATACTTCACTGCCCCAGATAAGGTTCCGATAAAAAGTTCTGCAGAC
    TAAATTTATTTCAGTCTCCTCTTCACCACCAAAATGCCCTCCTACGAAGCTCG
    AGCTAACGTCCACAAGTCCGCCTTTGCCGCTCGAGTGCTCAAGCTCGTGGCAG
    CCAAGAAAACCAACCTGTGTGCTTCTCTGGATGTTACCACCACCAAGGGGCCG
    CTCGAGCATGCATCTAGAGGGCCCAATTCGCCCTATAGTGAGTCGTATTACAA
    TTCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCC
    AACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAA
    GAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATG
    GACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAG
    CGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCC
    CTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGG
    CTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACT
    TGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTC
    GCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACT
    GGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTT
    GCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACG
    CGAATTTTAACAAAATTCAGGGCGCAAGGGCTGCTAAAGGAAGCGGAACACGT
    AGAAAGCCAGTCCGCAGAAACGGTGCTGACCCCGGATGAATGTCAGCTACTGG
    GCTATCTGGACAAGGGAAAACGCAAGCGCAAAGAGAAAGCAGGTAGCTTGCAG
    TGGGCTTACATGGCGATAGCTAGACTGGGCGGTTTTATGGACAGCAAGCGAAC
    CGGAATTGCCAGCTGGGGCGCCCTCTGGTAAGGTTGGGAAGCCCTGCAAAGTA
    AACTGGATGGCTTTCTTGCCGCCAAGGATCTGATGGCGCAGGGGATCAAGATC
    TGATCAAGAGACAGGATGAGGATCGTTTCGCATGATTGAACAAGATGGATTGC
    ACGCAGGTTCTCCGGCCGCTTGGGTGGAGAGGCTATTCGGCTATGACTGGGCA
    CAACAGACAATCGGCTGCTCTGATGCCGCCGTGTTCCGGCTGTCAGCGCAGGG
    GCGCCCGGTTCTTTTTGTCAAGACCGACCTGTCCGGTGCCCTGAATGAACTGC
    AGGACGAGGCAGCGCGGCTATCGTGGCTGGCCACGACGGGCGTTCCTTGCGCA
    GCTGTGCTCGACGTTGTCACTGAAGCGGGAAGGGACTGGCTGCTATTGGGCGA
    AGTGCCGGGGCAGGATCTCCTGTCATCCCACCTTGCTCCTGCCGAGAAAGTAT
    CCATCATGGCTGATGCAATGCGGCGGCTGCATACGCTTGATCCGGCTACCTGC
    CCATTCGACCACCAAGCGAAACATCGCATCGAGCGAGCACGTACTCGGATGGA
    AGCCGGTCTTGTCGATCAGGATGATCTGGACGAAGAGCATCAGGGGCTCGCGC
    CAGCCGAACTGTTCGCCAGGCTCAAGGCGCGCATGCCCGACGGCGAGGATCTC
    GTCGTGACCCATGGCGATGCCTGCTTGCCGAATATCATGGTGGAAAATGGCCG
    CTTTTCTGGATTCATCGACTGTGGCCGGCTGGGTGTGGCGGACCGCTATCAGG
    ACATAGCGTTGGCTACCCGTGATATTGCTGAAGAGCTTGGCGGCGAATGGGCT
    GACCGCTTCCTCGTGCTTTACGGTATCGCCGCTCCCGATTCGCAGCGCATCGC
    CTTCTATCGCCTTCTTGACGAGTTCTTCTGAATTGAAAAAGGAAGAGTATGAG
    TATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTC
    CTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAG
    TTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCT
    TGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTC
    TGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGT
    CGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGA
    AAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAA
    CCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCG
    AAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGA
    TCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCA
    CGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTA
    CTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGT
    TGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATA
    AATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCA
    GATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAAC
    TATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGC
    ATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAA
    CTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCAT
    GACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAG
    AAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGC
    TTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGA
    GCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAA
    ATACTGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTA
    GCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAG
    TGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATA
    AGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAG
    CGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGC
    CACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCG
    GAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTAT
    AGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTC
    GTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGT
    TCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCT
    GATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCG
    CAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGG

Claims (14)

The invention claimed is:
1. A retinol-producing host cell expressing a retinol dehydrogenase 22 (RDH22) homolog with at least about 30% identity to a polynucleotide according to SEQ ID NO: 1 or 4, wherein the host cell has been transformed with said polynucleotide encoding said RDH22 homolog and wherein the percentage of dihydroretinoids in a mix of retinoids produced using said host cell under suitable culture conditions is less than about 10% based on total retinoids.
2. The retinol-producing host cell according to claim 1, wherein said process furthermore comprises acetylation of retinol into retinyl acetate.
3. The retinol-producing host cell according to claim 2, wherein the percentage of retinyl acetate in said mix is at least about 80% based on total retinoids.
4. The retinol producing host cell according to claim 1, wherein the percentage of retinal in said mix is less than about 8% based on total retinoids.
5. The retinol-producing host cell according to claim 4, wherein the percentage of retinal is reduced to less than about 1% based on total retinoids.
6. The retinol producing host cell according to claim 1, wherein said mix of retinoids comprises trans and cis-isomers and wherein the percentage of trans-isomers is at least about 80% based on total cis and trans-isomers in said mix.
7. The retinol-producing host cell according to claim 1, wherein the RDH22 homolog is obtained from Yarrowia or Wickerhamomyces.
8. The retinol-producing host cell according to claim 1, furthermore expressing a retinol dehydrogenase 12 (RDH12) homolog.
9. Process for the production of a retinoid-mix comprising cultivation of a retinol-producing host cell according to claim 1 under suitable culture conditions.
10. Process according to claim 9, wherein said retinoid mix comprises dihydroretinal, dihydroretinol and dihydroretinyl acetate, retinol, retinal, retinyl acetate, and wherein the percentage of dihydroretinoids based on total retinoids is less than 10%.
11. Process according to claim 9, wherein the host cell is cultivated in the presence of a lipophilic solvent.
12. Process according to claim 11, wherein the retinoids are accumulated in the lipophilic solvent and optionally extracted from the lipophilic solvent.
13. Process according to claim 11, wherein the lipophilic solvent is selected from isoparaffins including mixtures of alkanes, cycloparaffin, isoalkanes, cycloalkanes, or dodecanes.
14. Process according to claim 9, wherein the host cell is a fungal host cell.
US18/700,931 2021-10-19 2022-10-19 Retinoid production Pending US20250002873A1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
US18/700,931 US20250002873A1 (en) 2021-10-19 2022-10-19 Retinoid production

Applications Claiming Priority (5)

Application Number Priority Date Filing Date Title
US202163257222P 2021-10-19 2021-10-19
EP21204854.0 2021-10-26
EP21204854 2021-10-26
US18/700,931 US20250002873A1 (en) 2021-10-19 2022-10-19 Retinoid production
PCT/EP2022/079143 WO2023067030A1 (en) 2021-10-19 2022-10-19 Retinoid production

Publications (1)

Publication Number Publication Date
US20250002873A1 true US20250002873A1 (en) 2025-01-02

Family

ID=84360127

Family Applications (1)

Application Number Title Priority Date Filing Date
US18/700,931 Pending US20250002873A1 (en) 2021-10-19 2022-10-19 Retinoid production

Country Status (4)

Country Link
US (1) US20250002873A1 (en)
EP (1) EP4419703A1 (en)
KR (1) KR20240099282A (en)
WO (1) WO2023067030A1 (en)

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP7472427B2 (en) 2017-09-25 2024-04-23 ディーエスエム アイピー アセッツ ビー．ブイ． Retinoid biosynthesis
WO2023214072A2 (en) * 2022-05-05 2023-11-09 Dsm Ip Assets B.V. Novel process

Family Cites Families (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7851199B2 (en) 2005-03-18 2010-12-14 Microbia, Inc. Production of carotenoids in oleaginous yeast and fungi
EP2078092A2 (en) 2006-09-28 2009-07-15 Microbia, Inc. Production of carotenoids in oleaginous yeast and fungi
CN105339490A (en) 2012-12-20 2016-02-17 帝斯曼知识产权资产管理有限公司 Acetyl transferases and their use for producing carotenoids
EP3286302A1 (en) 2015-04-21 2018-02-28 DSM IP Assets B.V. Geranylgeranyl pyrophosphate synthase
WO2019058001A1 (en) 2017-09-25 2019-03-28 Dsm Ip Assets B.V. Production of retinyl esters
JP7513520B2 (en) * 2017-09-25 2024-07-09 ディーエスエム アイピー アセッツ ビー．ブイ． Retinol Production
WO2019057999A1 (en) 2017-09-25 2019-03-28 Dsm Ip Assets B.V. Production of trans-retinal
JP7472427B2 (en) 2017-09-25 2024-04-23 ディーエスエム アイピー アセッツ ビー．ブイ． Retinoid biosynthesis
EA202191822A1 (en) 2018-12-31 2021-11-16 ДСМ АйПи АССЕТС Б.В. NEW ACETYL TRANSFERASE
EP3999634A1 (en) 2019-07-16 2022-05-25 DSM IP Assets B.V. Novel beta-carotene oxidases
JP2023507891A (en) 2019-12-30 2023-02-28 ディーエスエム アイピー アセッツ ビー．ブイ． Lipase modified strain

Also Published As

Publication number Publication date
KR20240099282A (en) 2024-06-28
WO2023067030A1 (en) 2023-04-27
EP4419703A1 (en) 2024-08-28

Similar Documents

Publication Publication Date Title
US12252724B2 (en) Biosynthesis of retinoids
JP7443656B2 (en) Production of retinyl esters
US20220064607A1 (en) Novel acetyl-transferases
EP4085132A1 (en) Lipase-modified strain
EP4237574A1 (en) In situ two-phase extraction system
EP4237570A1 (en) Fermentative production of isoprenoids
US20220356503A1 (en) Novel beta-carotene oxidases
US20250002873A1 (en) Retinoid production
US11905542B2 (en) Production of retinyl esters
WO2022003130A2 (en) Yeast expression system
WO2024160712A1 (en) Novel acetyl-transferases
WO2023006851A1 (en) Fermentative production of retinyl acetate in the presence of ethanol
EP4352243A1 (en) Method to produce retinyl acetate
CN118119700A (en) Retinoid Production
EP4658769A1 (en) Novel mutants of acetyl-transferase sb-atf
KR20250140088A (en) CARRP enzyme variants and their use in carotenoid and apocarotenoid production
WO2023006179A1 (en) Method to produce retinyl acetate
EP4189079A1 (en) Reduction of fatty acid retinyl ester formation

Legal Events

Date Code Title Description
AS Assignment

Owner name: DSM IP ASSETS B.V., NETHERLANDS

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:VYAS, VALMIK KANUBHAI;HOUSTON, PETER LOUIS;MCMAHON, JENNA;AND OTHERS;REEL/FRAME:067088/0779

Effective date: 20221019

STPP Information on status: patent application and granting procedure in general

Free format text: DOCKETED NEW CASE - READY FOR EXAMINATION